A simulated four-stage graphene distributed amplifier achieved up to 4 dB gain and 3.5 GHz bandwidth, which could be realized with future IC processes. A PCB level graphene distributed amplifier was fabricated as a proof of circuit concept.Ever since its discovery, graphene bears great expectations in high frequency electronics due to its irreplaceably high carrier mobility. However, it has long been blamed for the weakness in generating gains, which seriously limits its pace of development. Distributed amplification, on the other hand, has successfully been used in conventional semiconductors to increase the amplifiers’ gain-bandwidth product. In this paper, distributed amplification is first applied to graphene. Transmission lines phase-synchronize paralleled graphene field-effect transistors (GFETs), combining the gain of each stage in an additive manner. Simulations were based on fabricated GFETs whose  Their work achieved S21 of 10 at 1 GHzet al. fabricated graphene voltage amplifiers in complementary push-pull configuration, which generated a voltage gain of 11.4 dB and a −3 dB bandwidth of only 70 kHzHowever, the overall progress in electronics is greatly hindered by graphene’s gapless band structure1et al. and the authors’ group have proposed the multi-layer-routing inverted process for graphene integration, which serves as the foundation for future novel graphene circuit architectures1219On the other hand, graphene’s IC process has witnessed substantial improvements1219gm of each transistor in a phase-synchronized manner. Therefore, the bandwidth-gain-product could be increased. While the gain of a conventional cascade amplifier is the product of the gain of each stage, distributed amplifiers’ gain is directly proprotional to the number of stages. This trait makes distributed amplification scheme especially suitable for graphene, as each GFET stage generates very modest or less-than-unity gains. The otherwise product of the gain of each stage would be mediocre.In this paper, the graphene distributed amplifer is proposed to solve its long-standing weakness in amplification. Engineers have successfully applied distributed amplication to conventional semiconductor technologies to increase the amplifiers’ gain-bandwidth-product232425fT ranging from 8.2 GHz to 10.6 GHz and fmax 12.4 GHz to 16.6 GHz without de-embedding . The fmax exceeded fT in these works due the low resistance of the buried gatesFour GFETs with gate lengths of 300 nm through 500 nm have been fabricated, which generated fmax point of view. The GFETs employed a two-finger structure with each finger 6 μm wide. The gate dielectric employed HfO2 with equivalent oxide thickness (EOT) of 2 nm formed directly on the buried gate. Graphene was synthensized by CVD method and transferred by “bubbling” method as previously reported (details in Method)27GFETs in buried-gate structure have been fabricated. Cross-section and top views of a 400 nm-gate-length GFET structure are shown in 3N42O3fT/fmax of 8.2 GHz/16 GHz, 8.4 GHz/12.5 GHz, 9.1 GHz/12.4 GHz, 10.6 GHz/16.6 GHz, respectively, as shown in The probing pads were 80 μm × 80 μm with 100 μm pitch in ground-signal-ground (GSG) layout, as shown in d, Cg, Ld and Lg, constitute the gate and drain artificial lines. R1 and R2 form the two terminating resistances.1, R2, and the lumped passive components, Cg, Cd, Lg, Ld (details in Method). Source and load impedances were set as R1 and R2, respectively. Simulation #1 achieved S21 of roughly 4 dB and 3.5 GHz bandwidth, as shown in 1, R2, Cg, Cd, Lg and Ld resulted in 1.28 kΩ, 78.5 Ω, 0.01 pF, 0.7 pF, 105 nH and 7.6 nH, respectively. Simultion #1 represents situations when GFETs are exactly identical (T1~4: GFET #4). After the demonstration of high-performance individual GFETs in many lab works, efforts in graphene electronics society should now emphasize reproducibility and reliability to support the potential mass production.Circuit simulation based on measured S-parameters of the GFETs was performed in a standard circuit simulator, Agilent Advanced Design System (ADS). In Simulation #1, T1 through T4 all employed GFET #4, respresenting ideal situations when GFETs are exactly identical. The optimization was performed with three goals: 1) S21 > 5 dB; 2) S11 < −10 dB; 3) S22 < −10 dB. Variables were the terminating resistances, R1, R2, Cg, Cd, Lg and Ld resulted in 1.28 kΩ, 58 Ω, 0.01 pF, 1.2 pF, 86 nH and 7.2 nH, respectively, as shown in Another simulation, Simulation #2, was launched closer to real circumstances. GFET #1 through #4 were assigned to T1 through T4, respectively. The setup was the same with Simulation #1, which generated S21 of 3 dB and 3.5 GHz bandwidth, as shown in Simulation #1 and #2 for the first time demonstrated wide-band graphene amplifiers in literature. Both simulations were based on measured S-paramenters of the GFETs, illustrating the feasibilty of wide-band graphene amplifiers with present averge-performance GFETs. The passive component were subjected to the following equation that guarantees the phase-synchronization requirement:g, Cd, Lg and Ld were 0.01 pF, 0.7 pF, 105 nH and 7.6 nH, respectively. Miller effects obscured Cdrain and the gate oxide capacitance was roughly 0.08 pF, caculated according to the dimension and gate dielectric of the 400 nm GFET. Cgate was further decreased due to quantum capacitance effect321 and R2, which roughly equaled characteristic impedences of the gate and drain transmission lines, respectively, caculated as where C and L represents the capacitance and inductance at each node. For Simulation #1, Cg in Simulation #1 and #2.On the other hand, efforts have been made in this work to realize a PCB-level graphene distributed amplifier, which meets problems such as precise GFET models, the choice of discrete lumped passive components, and bonding wires. Any variations are likely to destroy the phase-synchronization between the gate and drain transmission lines, especially when any of the passive components values are too small, e.g., Cg, Cd, Lg, Ld are avoided in the simulations, Simulation #3 and #4. In particular, Cg was fixed at 1 pF . The terminating impedances were both set at 50 Ω. Simulation #3 used Cg = Cd = 1 pF and R1 = R2 = 50 Ω. The simulation result is shown in g, the gain decreased to −10~−5 dB and the bandwidth remained at 3 GHz. Simulation #4 further avoided matching sections at the input and output terminals by employing 50 Ω source and load impedence. Cg and Cd were 1 pF, and Lg and Ld 10 nH, which are common discrete devices. The simulation result is demonstrated in Firstly, small values for any of CA PCB-level graphene distributed amplifier was fabricated according to Simulation #4, which featured approximately 5 cm × 3 cm dimension, as shown in g. It increases with each GFET’s transconductance. The charateristic impedences of the artificial transmission lines are approximated as Test results are shown in Even though the fabricated work somewhat reassembles a real amplifier, future graphene distributed amplifers should be realized in IC technologies, where precise models of GFETs and passive components should be addressed. Besides, on-chip waveguides could be considered to replace artifical transmission lines. They might further reduces losses.Distributed amplifiers over the past decades have successfully been applied in compound semiconductor and CMOS technologies2324254/H2 flow rates were set at 4.5/500 sccm. After growth, Pt foils were quickly pulled out of the high temperature area27Graphene in this work was synthesized by CVD method on Pt foils as previously reported. Large scale monolayer graphene films were grown on 180 μm thick Pt foils  by ambient-pressure chemical vapor deposition (APCVD) method. The growth temperature was 1000 °C and CH2 with equivalent oxide thickness (EOT) of 2 nm was deposited as the gate oxide by atomic layer deposition (ALD) method. After graphene transfer, the channel of the GFETs was defined by contact photolithography and the residual graphene was removed by oxygen plasma etching. The source/drain contact was defined by electron-beam-lithography (EBL). Due to misalignment, certain source-gate and drain-gate overlaps existed in this patch of fabrication. The contacts were formed by lift-off process. The source/drain regions underwent 5 min UVO treatment before the sputtering of 40 nm Pt. The high work function of W induced more doping to graphene and UVO treatment enhanced the metal-graphene’s binding to each other. TML pattern characterization showed the lowest contact resistance of 500 Ωμm.The fabrication was based on 200 mm CMOS platform. Conventional BEOL processes fabricated buried gate/source/drain structures made of W. HfOThe Advanced Design System software was version 2012.08. In optimization toolset, the simulated anealing algorithm was used. Three goals were typically used: 1) S21 > 5 dB; 2) S11 < −10 dB; 3) S22 < −10 dB, in the frequency span from 500 MHz to 3 GHz. The weight of the each goals could be independently swiched.How to cite this article: Lyu, H. et al. Graphene Distributed Amplifiers: Generating Desirable Gain for Graphene Field-Effect Transistors. Sci. Rep.5, 17649; doi: 10.1038/srep17649 (2015).


Sarcoidosis is a multisystem inflammatory disorder of unknown cause. It most commonlyaffects the pulmonary system but can also affect the musculoskeletal system, albeitless frequently. In patients with sarcoidosis, rheumatic involvement is polymorphic.It can be the presenting symptom of the disease or can appear during its progression.Articular involvement is dominated by nonspecific arthralgia, polyarthritis, andLöfgren's syndrome, which is defined as the presence of lung adenopathy, arthralgia(or arthritis), and erythema nodosum. Skeletal manifestations, especially dactylitis,appear mainly as complications of chronic, multiorgan sarcoidosis. Muscle involvementin sarcoidosis is rare and usually asymptomatic. The diagnosis of rheumaticsarcoidosis is based on X-ray findings and magnetic resonance imaging findings,although the definitive diagnosis is made by anatomopathological study of biopsysamples. Musculoskeletal involvement in sarcoidosis is generally relieved withnonsteroidal anti-inflammatory drugs or corticosteroids. In corticosteroid-resistantor -dependent forms of the disease, immunosuppressive therapy, such as treatment withmethotrexate or anti-TNF-α, is employed. The aim of this review was to present anoverview of the various types of osteoarticular and muscle involvement insarcoidosis, focusing on their diagnosis and management. Rheumatic manifestations of sarcoidosis, although rare, includeinflammatory arthritis, periarticular soft tissue swelling, tenosynovitis, dactylitis,bone involvement, sarcoid myopathy, and bone loss. The primary types of articularinvolvement are Löfgren's syndrome and acute polyarthritis, whereas bone involvement isdominated by sarcoid dactylitis and osteolysis. Muscle involvement often goesunrecognized and can appear as chronic myopathy, acute myositis, or pseudotumor.Sarcoidosis can also manifest as calcium balance disturbances, principallyhypercalcemia, which is often asymptomatic but can occasionally be the presentingclinical symptom of the sarcoidosis.Sarcoidosis is a granulomatous disease of unknown etiology that involves multiplesystems. It most commonly affects the lungs, lymph nodes, skin, and eyes but can alsoaffect other organs and systems, including the musculoskeletal system. Treatment of rheumatic involvement often requires the use of nonsteroidalanti-inflammatory drugs (NSAIDs), corticosteroids, or methotrexate. Biological therapiessuch as the use of anti-TNF-α agents and anti-CD20 monoclonal antibodies have been shownto be effective in some cases of severe or refractory sarcoidosis. The aim of this review was to present an overview of the various types ofmusculoskeletal involvement in sarcoidosis, focusing on their diagnosis andmanagement.The diagnosis of sarcoidosis is based on clinical and radiological findings, togetherwith evidence of noncaseating granulomas in biopsy specimens, after other granulomatousdisorders, such as tuberculosis, have been excluded. Because of clinical and histological similarities with mycobacterial andfungal diseases, infectious causes have been investigated. However, such studies arecontroversial. Recent evidence suggests that a genetic component is implicated insusceptibility to sarcoidosis. There is a strong link between sarcoidosis and variantsin the class I and II HLA locus. A recent study identified annexin A11 as a novelnon-HLA susceptibility locus for sarcoidosis. Many other loci encoding TNF-α and co-stimulatory molecules onantigen-presenting cells such as CD80 and CD86, as well as the chemokine receptors CCR2and CCR5, have been found to increase susceptibility to sarcoidosis.The exact cause of sarcoidosis remains unknown. The Th1-type of inflammation is presentin the sarcoid granuloma which expresses and produces a variety of inflammatorycytokines, such as IL-2, IL-12, IL-6, and IFN-γ, as well as TNF-α, which is the centralmediator of this inflammatory process. Nonspecific arthralgia affects the majority of sarcoidosis patients,especially females. With the exception of Löfgren's syndrome, joint manifestations arerarely seen at symptom onset in sarcoidosis. Two types of arthritis, differing in theirclinical course and prognosis, have been identified. The first is acute polyarthritis,which is typically accompanied by erythema nodosum and occasionally by acute uveitis.Acute polyarthritis resolves without permanent sequelae. The second type is chronicsarcoid arthritis, which, although less common, can progress to joint deformity. Otherforms of articular manifestations, such as periarticular soft tissue swelling andtenosynovitis, can also be seen.The reported prevalence of arthritis in sarcoidosis ranges from 10% to 38%.8 Nons The most common form of acute arthropathy in sarcoidosis is Löfgren'ssyndrome, which occurs in acute onset sarcoidosis and typically manifests asbilateral hilar lymphadenopathy, arthritis, and erythema nodosum. Löfgren's syndromeis associated with a good prognosis and spontaneous remission. In patients with sarcoidosis, acute polyarthritis most commonly involvesthe ankles (in > 90% of cases), often bilaterally, followed by other large jointsof the lower limbs, only occasionally involving the small joints of hands and feet.This type of polyarthritis is only mildly painful, migratory and transient.Oligoarthritis or monoarthritis are relatively rare forms of acute sarcoidarthropathy.Acute polyarthritis occurs in 40% of patients with sarcoidosis, particularly in theearlier stages of the disease, and can be the presenting feature. It isself-limiting, is usually symmetric, and resolves without permanentsequelae. Various forms of chronic arthritis can occur in patients withsarcoidosis: nondeforming arthritis with granulomatous synovitis; Jaccoud'sarthropathy; and joint swelling adjacent to a sarcoid bone lesion. Among suchpatients, the arthritis is rheumatoid factor-positive in 10-47% of cases. Thatnonspecific reactivity is due to increased circulating polyclonal IgG. Therefore,sarcoidosis-related arthritis can mimic rheumatoid arthritis, especially whenaccompanied by joint deformities. The differential diagnosis is usually made on thebasis of clinical criteria, including negative serology for anti-cyclic citrullinatedpeptide antibodies and antinuclear antibodies, as well as the absence of the specificerosive joint deformity seen in rheumatoid arthritis.  A finding of granuloma on synovial biopsy helps in establishing thediagnosis of sarcoidosis. Although all joints can be affected, affected anklesstrongly indicate the diagnosis of sarcoidosis. In some cases, X-rays show softtissue swelling. Magnetic resonance imaging (MRI) can depict lesions that cannot bevisualized on X-rays.Chronic arthropathy is rare in sarcoidosis, occurring in only 0.2% of cases. It mostoften affects black males and is usually accompanied by other systemic disorders,mainly those of the lungs and eyes. Although tenosynovitis, tendinitis, bursitis, and synovitis can bedemonstrated on MRI scans, they are nonspecific findings and biopsy is thereforerequired in order to confirm the diagnosis of sarcoidosis.Among patients with sarcoidosis, tenosynovitis is common in the tendons of the anklesand wrist, occasionally accompanied by carpal tunnel syndrome in the lattercase. Sacroiliitis can reveal ankylosing spondylitis that can be associatedwith sarcoidosis, especially in patients testing positive for HLA-B27.Sacroiliac involvement in sarcoidosis is rare and generally unilateral. Sarcoidosiscannot be established without a biopsy to rule out tuberculosis or other infectiousprocess of that joint. The use of other immunosuppressive agents should be reserved forpatients with progressive chronic sarcoid arthropathy that is refractory to treatmentwith systemic corticosteroids or in whom steroids have generated side effects.According to the Brazilian Thoracic Association Guidelines for Interstitial LungDiseases, there are alternative treatments, such as methotrexate, azathioprine,leflunomide and hydroxychloroquine. Methotrexate is an efficient andcorticosteroid-sparing therapeutic agent for the treatment of musculoskeletalmanifestations of sarcoidosis. Many studies have shown the importance of TNF-α in sarcoid granulomadevelopment, which makes TNF-α a potential target in the treatment of sarcoidosis.Many interesting reports suggest some efficacy of TNF-α antagonists  in refractory sarcoidosis with musculoskeletalinvolvement.(-) Paradoxical cases of proven sarcoidosis have been reported in patientsreceiving anti-TNF-α agents for other chronic inflammatory rheumatic diseases. Thisparadoxical effect of anti-TNF-α agents must be known by the clinician. The use of B-cell-depleting agents might also be of benefit in sarcoidarthritis. A recent case report of a patient with sarcoidosis of the lungs and jointsshowed that rituximab is effective in treating sarcoidosis without major sideeffects. Based on the success of rituximab in this disease, other B-celltherapies, such as ocrelizumab, need to be evaluated in systemicsarcoidosis.In 90% of cases, acute polyarthritis resolves spontaneously. In others, it requirestreatment with NSAIDs, corticosteroid injections into the joint, or a short course ofcorticosteroids at 10-15 mg/day. Hydroxychloroquine and colchicine can be used insome cases, especially in those of Löfgren's syndrome.Bone involvement is reported in 1-15% of sarcoidosis patients. It is more common inblack patients and is usually accompanied by infiltrative skin lesions, especially lupuspernio.Although bone lesions are frequently asymptomatic, some sarcoidosis patients presentwith symptomatic dactylitis. Bone, skin and soft tissue are involved, especially inthe second and third phalanges, resulting in sausage-like fingers resembling thoseseen in the spondyloarthropathies. The bony lesions are usually cystic, sclerotic lesions rarely being reported.Multiple cystic lesions sometimes result in a "lacy" pattern, which is typical ofsarcoid bone disease. The classic lesions in the small bones of the hands and feetare known as Perthes disease and Jüngling's disease. They are well characterized onstandard X-rays.There are three radiological types of sarcoid bone disease: type I, characterized bybig cystic lesions , which iInvolvement of the axial skeleton and long bones is uncommon in sarcoidosis.Vertebral sarcoidosis can present as purely lytic lesions, as purely scleroticlesions (in rare cases) mimicking blastic metastases, or as a mixture of the two. Thelower dorsal and upper lumbar vertebrae are mostly involved. Because it can guide theselection of biopsy sites, MRI has gained attention as a modality that facilitatesthe histopathological confirmation of the diagnosis and can be used in evaluating theefficacy of the treatment of bone lesions. The MRI findings are nonspecific; showingmultifocal lesions that are hypointense on T1-weighted images and hyperintense onT2-weighted images.Any bone, including the skull, ribs, nasal bone, and long bones, can be affected insarcoidosis. In the skull, sarcoidosis manifests as asymmetrical, asymptomatic lyticlesions of variable size. Positron emission tomography/computed tomography (PET/CT)imaging can be useful in the assessment of bone involvement in sarcoidosispatients. Methotrexate and hydroxychloroquine can also be used. Although there issome evidence that anti-TNF-α agents are efficacious in sarcoid bone lesions, thiseffect needs to be verified.Asymptomatic osseous sarcoidosis generally does not require therapy, although theindications for therapeutic intervention are not well defined. However, treatment isusually indicated when the symptoms include uncontrolled pain, stiffness, or bonydestruction. Therapy generally consists of oral corticosteroids at 15-20 mg/day. Thedosage is adjusted according to the clinical response. The histological pattern of sarcoid myopathy is as a noncaseating granulomain the perimysial connective tissue. Large granulomas compress and destroy adjacentmuscle fibers, resulting in degeneration and focal lymphocyte infiltration, and foci ofnecrosis or fibrosis can also be observed. Fiber destruction in this disease is caused mainly by fiber infiltrationrather than by mechanical compression or ischemia.Muscle sarcoidosis is a rare entity and is usually asymptomatic. It is symptomatic inonly 1% of cases. It typically appears as a complication of systemicsarcoidosis. The cause is not only sarcoid myopathy but also the high circulating levelsof inflammatory cytokines such as TNF-α, IL-6, and IFN-γ.In a patient with sarcoidosis, the presence of muscle weakness, muscle pain, or musclenodules is suggestive of sarcoid myopathy. Fatigue and general weakness are common,which could explain why patients with sarcoidosis frequently experience exerciseintolerance.: chronic myopathy (seen in 86% of cases), which is the most common form,characterized by an insidious onset of proximal muscle weakness with normal or elevatedserum levels of muscle enzymes; acute myositis (seen in 11% of cases); and nodular ortumor-like myositis (seen in only 3% of cases). Nodular myopathy manifests as multiple,tumor-like, palpable nodules in the muscles. The use of MRI and PET/CT facilitates the diagnosis of musclesarcoidosis.In sarcoid myopathy, three clinical patterns are generally recognized37: ch Thalidomide and infliximab have been found to be beneficial in some casesof sarcoid myopathy. The effectiveness of these medications seems related to TNF-αinhibition. Corticosteroid-induced myopathy can also occur as a complication of thetreatment of sarcoidosis. Affected patients typically develop proximal muscle weaknessthat has a gradual onset  and is accompanied by muscle wasting. Acommon manifestation is difficulty getting up from a chair or climbing stairs. Myalgiaand muscle tenderness are not observed.The mainstay of the treatment of patients with muscle sarcoidosis is 8-12 weeks ofsystemic glucocorticoid therapy at an initial daily dose of 0.5-1 mg/kg with progressivetapering. Methotrexate, chloroquine and azathioprine have been used incorticosteroid-resistant and corticosteroid-dependent forms. The use of anti-TNF-α agents should be avoided in patients who have activeSLE. In rare cases, sarcoidosis and Sjögren's syndrome can both affect the salivaryglands. Dryness and diffuse swelling of oral mucosal tissues can be the presentingsymptom of sarcoidosis.Sarcoidosis can be associated with other chronic inflammatory disease like systemiclupus erythematosus (SLE), Sjögren's syndrome or psoriatic arthritis. In sarcoidosis,suspicion of SLE is raised when the patient develops a butterfly rash or discoidlesions. The treatment of sarcoidosis patients with SLE is challenging and should beindividualized. Although anti-TNF-α therapy is helpful in psoriasis, it can alsoparadoxically induce progressive psoriasis, and patients treated with anti-TNF-α agentsshould be closely monitored.Approximately 6% of all patients with sarcoidosis develop a psoriatic form of arthritis. The high levels of calcitriol induce osteoclast activation and boneresorption. In addition, corticosteroid-treated patients are at a higher risk ofosteoporosis. Bone loss could be also increased by high levels of parathyroidhormone-related peptide identified in sarcoid tissue. Corticosteroids have been successfully used to improve disorders of calciummetabolism.  Mycophenolate mofetil and infliximab have been used in selectcases.In sarcoidosis, hypercalciuria is more common than is hypercalcemia. Either can becaused by nephrocalcinosis, kidney stones, or renal failure. Granulomatous macrophagesincrease conversion of 25-hydroxyvitamin D to active 1,25-dihydroxyvitamin D, leading to increased calcium absorption by the intestine. The bone loss in sarcoidosis can be caused by multiple factors, including diffuseskeletal granulomatosis, calcitriol, osteoclast activating factor, and glucocorticoidtherapy, particularly in postmenopausal patients. In one study of corticosteroid therapyin patients with sarcoidosis, the authors found that the rate of bone loss incorticosteroid-treated postmenopausal patients with sarcoidosis was greater than thatreported for corticosteroid-treated patients with rheumatoid arthritis orasthma. Guidelines published by the American College of Rheumatology recommend thatpatients receiving corticosteroids should undergo bone mineral densitytesting. The World Health Organization fracture assessment risk tool can be used inorder to calculate patient risk of fracture.The prevention and treatment of bone loss in patients with sarcoidosis is difficult.Calcium and vitamin D, both commonly administered to patients at risk for osteoporosis,should be considered with caution in patients with hypercalcemia, hypercalciuria, highlevels of parathormone, and kidney stones. Further studies on bisphosphonate use in osteoporosis during sarcoidosisare needed.For patients with sarcoidosis at risk of developing osteoporosis because of prolongeduse of corticosteroids, bisphosphonates are effective in preventingglucocorticoid-induced bone loss. The American College of Rheumatology recommends usingbisphosphonates in low-risk patients receiving corticosteroids at doses of = 7.5 mg/day,as well as in all medium- or high-risk patients receiving corticosteroids. Of theavailable bisphosphonates, alendronate, risedronate, zoledronic acid, and teriparatideeffectively reduce bone loss and can thus diminish fracture risk.Although sarcoidosis can affect any organ, sarcoidosis involving the musculoskeletalsystem is rare. The disease can affect the muscles, joints and the bones. Thoseconditions, which are polymorphic, can be the presenting symptoms the disease or canappear during the course of its progression. Corticosteroids are the cornerstone ofsarcoidosis treatment but only have a postponing effect. Prospective, randomized,controlled trials assessing anti-TNF-α agents are needed in order to evaluate theirefficacy in cases of sarcoidosis with rheumatic complications.  As manifestações reumáticas da sarcoidose, embora raras, incluem artriteinflamatória, edema de partes moles periarticulares, tenossinovite, dactilite,acometimento ósseo, miopatia sarcoide e perda óssea. Os principais tipos deacometimento articular são síndrome de Löfgren e poliartrite aguda, enquanto oacometimento ósseo é dominado por dactilite sarcoide e osteólise. O acometimentomuscular muitas vezes passa despercebido, podendo aparecer como miopatia crônica,miosite aguda ou pseudotumor. A sarcoidose também pode se manifestar como distúrbiosno equilíbrio de cálcio, principalmente hipercalcemia, que muitas vezes éassintomática, mas pode ocasionalmente ser o sintoma clínico de apresentação dasarcoidose.A sarcoidose é uma doença granulomatosa de etiologia desconhecida que acomete váriossistemas. Afeta mais frequentemente pulmões, linfonodos, pele e olhos, mas tambémpode afetar outros órgãos e sistemas, incluindo o sistemamúsculo-esquelético. O tratamento do acometimento reumático muitas vezes requer o uso deanti-inflamatórios não esteroidais (AINEs), corticosteroides ou metotrexato. Asterapias biológicas como o uso de agentes anti-TNF-α e anticorpos monoclonaisanti-CD20 têm se mostrado eficazes em alguns casos de sarcoidose grave ourefratária. O objetivo desta revisão foi apresentar uma visão geral dos vários tiposde acometimento músculo-esquelético na sarcoidose, com foco no diagnóstico emanejo.O diagnóstico de sarcoidose baseia-se em achados clínicos e radiológicos, juntamentecom evidências de granulomas não caseosos em espécimes de biópsia, após outrosdistúrbios granulomatosos, como a tuberculose, terem sido excluídos. Em virtude de semelhanças clínicas e histológicas com doençasmicobacterianas e fúngicas, causas infecciosas têm sido investigadas. Porém, taisestudos são controversos. Evidências recentes sugerem que um componente genético está implicado nasuscetibilidade à sarcoidose. Há uma forte ligação entre a sarcoidose e variantes nolócus HLA de classe I e II. Um estudo recente identificou a anexina A11 como um novolócus não-HLA de suscetibilidade para a sarcoidose. Muitos outros locos que codificam TNF-α e moléculas coestimuladoras emcélulas apresentadoras de antígenos como CD80 e CD86, assim como os receptores dequimiocinas CCR2 e CCR5, mostraram aumentar a suscetibilidade àsarcoidose.A causa exata da sarcoidose permanece desconhecida. A inflamação do tipo Th1 estápresente no granuloma sarcoide que expressa e produz uma variedade de citocinasinflamatórias, como IL-2, IL-12, IL-6 e IFN-γ, assim como TNF-α, que é o mediadorcentral desse processo inflamatório. A artralgia inespecífica afeta a maioria dos pacientes com sarcoidose,especialmente os do sexo feminino. Excetuando-se a síndrome de Löfgren, manifestaçõesarticulares são raramente vistas no início dos sintomas na sarcoidose. Dois tipos deartrite, que diferem quanto à evolução clínica e ao prognóstico, foram identificados.O primeiro é a poliartrite aguda, que é tipicamente acompanhada de eritema nodoso eocasionalmente de uveíte aguda. A poliartrite aguda se resolve sem sequelaspermanentes. O segundo tipo é a artrite sarcoide crônica, que, embora menosfrequente, pode evoluir para deformidade articular. Outras formas de manifestaçõesarticulares, como o edema de partes moles periarticulares e a tenossinovite, tambémpodem ser vistas.A prevalência relatada de artrite na sarcoidose varia de 10% a 38%.8 A ar A forma mais frequente de artropatia acuda na sarcoidose é a síndromede Löfgren, que ocorre na sarcoidose de início agudo e tipicamente se manifestacomo linfadenopatia hilar bilateral, artrite e eritema nodoso. A síndrome deLöfgren está associada a bom prognóstico e remissão espontânea. Em pacientes com sarcoidose, a poliartrite aguda acomete maisfrequentemente os tornozelos (em > 90% dos casos), muitas vezes bilateralmente,seguidos de outras grandes articulações dos membros inferiores, acometendo apenasocasionalmente as pequenas articulações das mãos e dos pés. Esse tipo depoliartrite é apenas levemente doloroso, migratório e transitório. Oligoartrite oumonoartrite são formas relativamente raras de artropatia sarcoide aguda.A poliartrite aguda ocorre em 40% dos pacientes com sarcoidose, particularmentenos estágios iniciais da doença, e pode ser a característica de apresentação. Éautolimitada, geralmente simétrica e se resolve sem sequelaspermanentes. Várias formas de artrite crônica podem ocorrer em pacientes comsarcoidose: artrite não deformadora com sinovite granulomatosa; artropatia deJaccoud; e edema articular adjacente a uma lesão sarcoide do osso. Entre taispacientes, a artrite é positiva para fator reumatoide em 10-47% dos casos. Essareatividade inespecífica é devida ao aumento de IgG policlonal circulante.Portanto, a artrite por sarcoidose pode simular a artrite reumatoide,especialmente quando acompanhada de deformidades articulares. O diagnósticodiferencial geralmente é feito com base em critérios clínicos, incluindo sorologianegativa para anticorpos anti-peptídeos citrulinados cíclicos e para anticorposantinucleares, assim como a ausência da deformidade articular erosiva específicavista na artrite reumatoide. O achado de granuloma na biópsia sinovial ajuda a estabelecer odiagnóstico de sarcoidose. Embora todas as articulações possam ser afetadas,tornozelos afetados indicam fortemente o diagnóstico de sarcoidose. Em algunscasos, as radiografias mostram edema de partes moles. A ressonância magnética (RM)é capaz de revelar lesões que não podem ser visualizadas nas radiografias.A artropatia crônica é rara na sarcoidose, ocorrendo em apenas 0,2% dos casos.Afeta mais frequentemente indivíduos negros do sexo masculino e é geralmenteacompanhada de distúrbios sistêmicos, principalmente os distúrbios dos pulmões edos olhos. Embora a tenossinovite, a tendinite, a bursite e a sinovite possamser demonstradas em imagens de RM, são achados inespecíficos, e, portanto, abiópsia é necessária para a confirmação do diagnóstico de sarcoidose.Entre os pacientes com sarcoidose, a tenossinovite é frequente nos tendões dostornozelos e pulsos, ocasionalmente acompanhada de síndrome do túnel do carpo nosegundo caso.  A sacroileíte pode revelar espondilite anquilosante que pode estarassociada à sarcoidose, especialmente em pacientes HLA-B27-positivos.O acometimento sacroilíaco na sarcoidose é raro e geralmente unilateral. Asarcoidose não pode ser estabelecida sem uma biópsia para excluir a tuberculose ououtros processos infecciosos dessa articulação. O uso de outros agentes imunossupressores deve ser reservado parapacientes com artropatia sarcoide crônica progressiva refratária a tratamento comcorticosteroides sistêmicos ou nos quais os esteroides geraram efeitos colaterais.De acordo com as Diretrizes de Doenças Pulmonares Intersticiais da SociedadeBrasileira de Pneumologia e Tisiologia, existem tratamentos alternativos, como o metotrexato, a azatioprina,a leflunomida e a hidroxicloroquina. O metotrexato é um agente terapêuticoeficiente e poupador de corticosteroides para o tratamento de manifestaçõesmúsculo-esqueléticas da sarcoidose. Muitos estudos demonstraram a importância do TNF-α no desenvolvimentodo granuloma sarcoide, o que faz do TNF-α um potencial alvo no tratamento dasarcoidose. Muitos relatos interessantes sugerem alguma eficácia de antagonistasdo TNF-α  na sarcoidose refratária comacometimento músculo-esquelético.(-) Casos paradoxais de sarcoidose comprovada foram relatados empacientes tratados com agentes anti-TNF-α para outras doenças reumáticasinflamatórias crônicas. Esse efeito paradoxal dos agentes anti-TNF-α deve serconhecido pelo clínico. É possível que o uso de agentes depletores de células B também possaser benéfico na artrite sarcoide. Um relato de caso recente de um paciente comsarcoidose dos pulmões e articulações demonstrou que o rituximabe é efetivo notratamento da sarcoidose, sem grandes efeitos colaterais. Com base no sucesso do rituximabe nessa doença, outras terapias comalvo em células B, como o ocrelizumabe, precisam ser avaliadas na sarcoidosesistêmica.Em 90% dos casos, a poliartrite aguda se resolve espontaneamente. Em outros,requer tratamento com AINEs, injeções de corticosteroides na articulação ou umcurso curto de 10-15 mg/dia de corticosteroides. A hidroxicloroquina e acolchicina podem ser usadas em alguns casos, especialmente nos de síndrome deLöfgren.O acometimento ósseo é relatado em 1-15% dos pacientes com sarcoidose. É maisfrequente em pacientes negros e é geralmente acompanhado de lesões cutâneasinfiltrativas, especialmente o lúpus pernio.Embora as lesões ósseas sejam frequentemente assintomáticas, alguns pacientes comsarcoidose apresentam dactilite sintomática. Osso, pele e tecido mole estãoenvolvidos, especialmente na segunda e terceira falanges, resultando em dedos emsalsicha que lembram aqueles vistos nas espondiloartropatias.As lesões ósseas geralmente são císticas, sendo raros os relatos de lesõesescleróticas. Múltiplas lesões císticas às vezes resultam em um padrão "rendado",que é típico da doença sarcoide do osso. As lesões clássicas nos pequenos ossosdas mãos e dos pés são conhecidas como doença de Perthes e doença de Jüngling. Sãobem caracterizadas nas radiografias padrão.Existem três tipos radiológicos de doença sarcoide do osso: tipo I, caracterizadopor grandes lesões císticas , queé bO acometimento do esqueleto axial e de ossos longos é infrequente na sarcoidose. Asarcoidose vertebral pode se apresentar como lesões puramente líticas, como lesõespuramente escleróticas (em casos raros) simulando metástases blásticas ou como umamistura das duas. As vértebras dorsais inferiores e lombares superiores sãoacometidas em sua maioria. Uma vez que a RM pode orientar a seleção de locais debiópsia, ela tem recebido atenção como modalidade facilitadora da confirmaçãohistopatológica do diagnóstico e pode ser usada na avaliação da eficácia dotratamento das lesões ósseas. Os achados da RM são inespecíficos, mostrando lesõesmultifocais que são hipointensas em imagens em T1 e hiperintensas em imagens emT2.Qualquer osso, incluindo o crânio, as costelas, o osso nasal e os ossos longos,pode ser afetado na sarcoidose. No crânio, a sarcoidose se manifesta como lesõeslíticas assimétricas assintomáticas de tamanho variado. positron emission tomography/tomografiacomputadorizada (PET/TC) podem ser úteis na avaliação de acometimento ósseo empacientes com sarcoidose.Imagens de O metotrexato e a hidroxicloroquina também podem ser usados. Emboraexistam evidências de que os agentes anti-TNF-α são eficazes nas lesões sarcoidesdo osso, esse efeito precisa ser verificado.A sarcoidose óssea assintomática em geral não requer terapia, embora as indicaçõespara intervenção terapêutica não estejam bem definidas. Porém, geralmente seindica tratamento quando os sintomas incluem dor não controlada, rigidez oudestruição óssea. A terapia em geral consiste em 15-20 mg/dia de corticosteroidesorais. A dosagem é ajustada de acordo com a resposta clínica. O padrão histológico da miopatia sarcoide é o de granuloma não caseosono tecido conjuntivo perimisial. Grandes granulomas comprimem e destroem as fibrasmusculares adjacentes, resultando em degeneração e infiltração linfocitária focal, efocos de necrose ou fibrose também podem ser observados. A destruição das fibras nessa doença é causada principalmente porinfiltração das fibras, em vez de por compressão mecânica ou isquemia.A sarcoidose muscular é uma entidade rara e geralmente assintomática. É sintomáticaem apenas 1% dos casos. Tipicamente aparece como complicação da sarcoidosesistêmica. A causa não é apenas a miopatia sarcoide, mas também os altos níveiscirculantes de citocinas inflamatórias como TNF-α, IL-6 e IFN-γ.Em um paciente com sarcoidose, a presença de fraqueza muscular, dor muscular ounódulos musculares é sugestiva de miopatia sarcoide. Fatiga e fraqueza geral sãocomuns, o que poderia explicar porque pacientes com sarcoidose frequentementeapresentam intolerância ao exercício.: miopatia crônica (vista em 86% dos casos), que é a forma mais comum,caracterizada por um início insidioso de fraqueza muscular proximal com níveisséricos de enzimas musculares normais ou elevados; miosite aguda (vista em 11% doscasos); e miosite nodular ou tumoral (vista em apenas 3% dos casos). A miopatianodular se manifesta como múltiplos nódulos palpáveis e tumorais nosmúsculos. O uso da RM e da PET/TC facilita o diagnóstico de sarcoidosemuscular.Na miopatia sarcoide, três padrões clínicos são geralmente reconhecidos37: mi A talidomida e o infliximabe se mostraram benéficos em alguns casos demiopatia sarcoide. A eficácia desses medicamentos parece estar relacionada à inibiçãodo TNF-α. A miopatia induzida por corticosteroide também pode ocorrer comocomplicação do tratamento da sarcoidose. Os pacientes afetados tipicamentedesenvolvem fraqueza muscular proximal de início gradual (ao longo de várias semanas)e acompanhada de perda muscular. Uma manifestação frequente é a dificuldade de selevantar de uma cadeira ou subir escadas. Mialgia e sensibilidade muscular não sãoobservadas.A base do tratamento de pacientes com sarcoidose muscular são 8-12 semanas de terapiacom glicocorticoide sistêmico na dose diária inicial de 0,5-1 mg/kg, com reduçãoprogressiva. O metotrexato, a cloroquina e a azatioprina têm sido usadas em formasresistentes ao corticosteroide ou corticosteroide dependentes. O uso de agentes anti-TNF-α deve ser evitado em pacientes que têm LESativo. Em raros casos, a sarcoidose e a síndrome de Sjögren podem ambas afetar asglândulas salivares. Secura e edema difuso dos tecidos das mucosas orais pode ser osintoma de apresentação da sarcoidose.A sarcoidose pode estar associada a outras doenças inflamatórias crônicas como olúpus eritematoso sistêmico (LES), a síndrome de Sjögren ou a artrite psoriásica. Nasarcoidose, a suspeita de LES é levantada quando o paciente desenvolve erupção emborboleta ou lesões discoides. O tratamento de pacientes com sarcoidose e LES édesafiador e deve ser individualizado. Embora a terapia anti-TNF-α seja útil na psoríase, paradoxalmente,também pode induzir psoríase progressiva, e pacientes tratados com agentes anti-TNF-αdevem ser monitorados cuidadosamente.Aproximadamente 6% de todos os pacientes com sarcoidose desenvolvem artritepsoriásica. Os altos níveis de calcitriol induzem a ativação de osteoclastos e areabsorção óssea. Além disso, pacientes tratados com corticosteroides apresentam ummaior risco de osteoporose. A perda óssea poderia ser também aumentada pelos altosníveis de peptídeo relacionado ao hormônio paratireoide identificados em tecidosarcoide. Os corticosteroides têm sido usados com sucesso para melhorar osdistúrbios do metabolismo do cálcio. O micofenolato de mofetil e o infliximabe têm sido usados em casosselecionados.Na sarcoidose, a hipercalciúria é mais frequente do que a hipercalcemia. Ambas podemser causadas por nefrocalcinose, pedras nos rins ou insuficiência renal. Macrófagosgranulomatosos aumentam a conversão da 25-hidroxivitamina D em 1,25-hidroxivitamina D, levando ao aumento da absorção de cálcio pelo intestino.A perda óssea na sarcoidose pode ser causada por vários fatores, incluindogranulomatose esquelética difusa, calcitriol, fator ativador de osteoclastos, eterapia com glicocorticoides, particularmente em pacientes na pós-menopausa. Em umestudo sobre corticoterapia em pacientes com sarcoidose, os autores observaram que ataxa de perda óssea em pacientes portadoras de sarcoidose na pós-menopausa quereceberam corticosteroide foi maior do que a relatada para pacientes portadores deartrite reumatoide ou asma que receberam corticosteroide. As diretrizes publicadas pelo American College ofRheumatology recomendam que pacientes que recebem corticosteroides devemser submetidos a testes de densidade mineral óssea. A ferramenta de avaliação de risco de fratura da Organização Mundial daSaúde pode ser usada para calcular o risco de fratura do paciente.A prevenção e o tratamento da perda óssea em pacientes com sarcoidose é difícil. Ocálcio e a vitamina D, ambos frequentemente administrados a pacientes com risco deosteoporose, devem ser considerados com cautela em pacientes com hipercalcemia,hipercalciúria, níveis elevados de paratormônio e pedras nos rins.American College ofRheumatology recomenda o uso de bifosfonados em pacientes de baixo riscoque recebem corticosteroides em doses = 7,5 mg/dia, assim como em todos os pacientesde médio ou alto risco que recebem corticosteroides. Dos bifosfonados disponíveis, oalendronato, o risedronato, o ácido zoledrônico e a teriparatida reduzem eficazmentea perda óssea e, assim, podem diminuir o risco de fratura. São necessários mais estudos sobre o uso de bifosfonados na osteoporosedurante a sarcoidose.Para pacientes com sarcoidose que apresentam risco de desenvolver osteoporose emrazão do uso prolongado de corticosteroides, os bifosfonados são eficazes naprevenção de perda óssea induzida por glicocorticoides. O Embora a sarcoidose possa afetar qualquer órgão, o acometimento do sistemamúsculo-esquelético é raro. A doença pode afetar os músculos, as articulações e osossos. Essas condições, que são polimórficas, podem ser os sintomas de apresentaçãoda doença ou podem aparecer no curso de sua progressão. Os corticosteroides são opilar do tratamento da sarcoidose, mas apenas têm efeito de adiamento. Sãonecessários ensaios prospectivos, randomizados e controlados para a avaliação de suaeficácia em casos de sarcoidose com complicações reumáticas.


In the race of gaining higher energy density, carbon’s capacity to retain power density is generally lost due to defect incorporation and resistance increment in carbon electrode. Herein, a relationship between charge carrier density/charge movement and supercapacitance performance is established. For this purpose we have incorporated the most defect-free pristine graphene into defective/sacrificial graphene oxide. A unique co-solvent-based technique is applied to get a homogeneous suspension of single to bi-layer graphene and graphene oxide. This suspension is then transformed into a 3D composite structure of pristine graphene sheets (GSs) and defective N-doped reduced graphene oxide (N-RGO), which is the first stable and homogenous 3D composite between GS and RGO to the best of our knowledge. It is found that incorporation of pristine graphene can drastically decrease defect density and thus decrease relaxation time due to improved associations between electrons in GS and ions in electrolyte. Furthermore, N doping is implemented selectively only on RGO and such doping is shown to improve the charge carrier density of the composite, which eventually improves the energy density. After all, the novel 3D composite structure of N-RGO and GS greatly improves energy and power density even at high current density (20 A/g). In supercapacitors, also known as double layer capacitors, the movement of charge determines both the energy and power densities. Unlike batteries, supercapacitors store their energy in electrode-electrolyte interphase generated due to the application of voltage bias12356782 g−1, is much higher than that of activated graphene oxide (GO) with surface area of 3100 m2 g−1 13et al.z orbital, and the fifth is an additional electron that actually occupies a π*-donor state, providing n-type doping to graphene. In contrast, pyridinic and pyrrolic nitrogen atoms are found near a carbon vacancy and so they have only two carbon atoms as neighbors. As a consequence, they contribute 2 electrons with only two σ bonds along with one electron in the pz orbital forming a π-bond with its neighbor, and thus leave two electrons as a lone pair. Imparting N-doping in carbon, can therefore have huge implication on its surface morphology as well as its electrochemical performance. Hence, it can be concluded that in carbon materials like graphene, one can achieve superior capacitance/energy density by simply increasing the charge carrier density as well as sheet exposure to electrolyte. Therefore, graphene can be an excellent candidate for obtaining higher energy density without significantly disrupting the power density.Charge storage and utilization in supercapacitor devices, are highly dependent on the defect density of the carbon. It has been reported that the volumetric capacitance of the supercapacitors gets saturated, if surface area of carbon increases beyond a certain limit, especially when pores have thin walls similar to the thickness of grapheneet al. showed a tremendous specific capacitance improvement after PANI incorporation in carbon aerogel. However the capacitance retention of the material was quite low and it retained only 66% of the initial capacity at 10 A/gHerein the major challenge is to incorporate N heteroatom into the graphene matrix as pristine graphene has a perfect structure, and it is difficult to introduce heteroatom without disrupting its lattice121719202122242526Herein, we take the advantage of π electronic interaction of graphitic carbon to obtain a stable uniform 3D composite of N- RGO and pristine GS via freeze drying for generation of high surface area and macropores. Giving a 3D structure to the composite is essential to exploit the complete surface of GSs. To obtain this 3D structure by freeze drying, it is of greatest importance to identify the solvent, which can disperse and keep both graphene and GO sheets stable and be compatible for freeze drying process as well. Therefore, the use of normal organic solvents, which are generally being used for dispersing graphite, is out of question as they can introduce other functional groups onto graphene sheets during processinga (HSP distance) curve shows the parabolic behavior with minimum at 40% ethanol concentration in water, which clearly illustrates that the adaptability of co-solvent system for graphite is maximum at this ethanol concentration. On the basis of cohesive energy, calculated by considering atomic and molecular bonding, such as dispersive δD, polar δP, and hydrogen bonding δH, . Therefore, the smaller the value of Ra, the better the adaptation of solvent for solute. For the present system on the basis of Ra value, we have tried to determine the exact concentration of ethanol and water in the co-solvent system, required for the efficient dispersion of graphite. For our present system, 40% of ethanol in water was found to give the best dispersion of graphite. The concentration of graphite in 40% ethanol-60% water system is calculated to be 0.32 mg/ml as shown in where RQuality of the dispersed graphene was further evaluated. Figure S2 of SI shows that dispersed sample mainly comprises of 1–2 layers of graphene. The electron diffraction pattern shows that the mixed solvent system does not disrupt the chemical integrity of the obtained graphene. From atomic resolution HR-TEM images, the lattice constant for graphene is found to be 0.25 nm, which is consistent with that of pristine graphene. On the other hand, Figure S3 of SI shows the dispersed GO morphology. It is seen that GO sheets are efficiently dispersed in water-ethanol azeotropic system like in pure water system.Topographical features of the freeze-dried N-RGO-GS composite and N-RGO are shown in 3234The effectiveness of present reduction process on the prepared composite material is analyzed by XPS analysis. 3g−1 and from 190 to 171 m2g−1, is observed after graphene sheet incorporation. However, micropore volume is found to increase in N-RGO-GS . Interestingly, very pronounced G band and 2D band are observed in N-RGO-GS in comparison to N-RGO, which clearly indicates the efficient incorporation of GSs into GO matrix. BET surface area of the prepared samples was also evaluated as shown in Figure S7 of SI. An obvious decrement in mesopores volume and surface area, from 0.79 to 0.51 cmN-RGO-GS . This vaThe electrochemical testing was performed in a three-electrode setup by keeping Pt as a counter and SCE as a reference electrode. The effect of GS addition on the electrochemical behavior can be easily seen from the CV performance in max of To understand this excellent stability in capacitance in N-RGO-GS as compared with N-RGO at higher current density, we have carried out the impedance analysis on the samples. As a result, from the lower relaxation time and higher frequency response of N-RGO-GS as compared with N-RGO, it can be understood that at lower frequency alternating current (AC) field, almost all the polarized ions in N-RGO are fully aligned, and hence N-RGO possesses better capacitance than N-RGO-GS. It is necessary to mention here that the defect density in N-RGO is much higher  as compared to N-RGO-GS, which enables much higher charge storage in this material at lower frequency/current density. However, as the AC frequency increases, most of the polarized ions in N-RGO cannot follow the higher frequency field at each direction reversal, due to poor conductivity of this sample as compared with its counterpart. As the high-frequency AC field changes direction faster, these ions “relax” to nonaligned positions, where N-RGO cannot store energy as much as N-RGO-GS can. Hence, a decrement in the N-RGO performance is observed at higher frequencyTo get a better picture of the origin of this phenomenon, Mott-Schottky plots are obtained for N-RGO and N-RGO-GS . Before 43444546In conclusions, herein, with the motive of finding a common ground of improving energy density without significantly losing the power density and proving the importance of defect-free conductive surface in supercapacitors, we have developed for the first time a novel uniform 3D high surface area structure of N-doped reduced graphene oxide–pristine graphene sheet (N-RGO-GS) composite, via simple freeze drying process, using a common ethanol-water azeotrope solvent. In the resultant novel 3D composite, the graphene sheets are present between two graphene oxide layers, and this kind of packing can keep the graphene sheets separate, thus offering efficient electrical transmission path in the composite scaffold with high surface area. The well-separated sheet structure provides much higher surface accessibility to the electrolyte ions, improving the electrolyte-electrode interaction. Furthermore, it was found that N-doping induces n-type charge carriers and helps in increasing the capacitance (energy density) of the N-RGO-GS. On the other hand, due to the incorporation of highly conductive pristine graphene, the defect density, which affects the frequency response of the capacitance, has been reduced, causing a drastic reduction in relaxation time. Therefore, much improved overall supercapacitance performance is observed even at higher current density (20 A/g), illustrating superior energy density retention. Through this study, we have also shown that in addition to higher surface area, a defect-free conductive surface is very essential for obtaining an excellent candidate for supercapacitor. This facile method of incorporating pristine graphene into disordered graphene oxide is expected to have huge implication towards the development of high efficient supercapacitor and battery materials in the energy storage field and can definitely open new dimensions in both synthetic and electro-chemistry of new carbon composite materials.4 in H2SO4:H3PO4 (9:1) solution. The obtained mixture was then stirred for 12 h at 50 °C. After getting brownish colour, 400 ml ice was added to the solution followed by dropwise addition of 3 ml H2O2. Obtained yellowish solution was then stirred for 2 h more. The final product was then obtained by centrifuging and washing with HCl and water. The obtained graphene oxide (GO) product was then dried at 70 °C overnight.The graphene oxide was synthesized using improved Hummers method. In brief 3.0 gm of graphite , was mixed with 18.0 gm of KMNOFirstly, 10 mg graphite powder was dispersed in 50 ml ethanol/water mixture with 40 vol% ethanol volume, using a bath sonicator operated at low power for 8 h continuously. The obtained dispersion was then centrifuged at 3000 rpm for 30 mins, and the supernatant was collected. On the other hand, the GO product was dispersed in the same azeotropic solvent (50 ml ethanol/water mixture with 40 vol% ethanol volume), by 2 h of sonication for 25 mg GO. The GO dispersant was then centrifuged at 4000 rpm for 45 mins to get stable GO solution. To obtain the final composite, both graphene and graphene oxide solutions are then mixed together under sonication for 30 mins. The obtained composite solution was then freeze-dried in a freezer (operated at −40 °C) and dryer (operated at 5 m torr) . The obtained graphene oxide-graphene sheet product is termed at GO-GS composite. For comparison, only GO dispersion was also freeze-dried by keeping all the conditions similar to those of GO-GS. In addition, we have tried to synthesize only 3D graphene by keeping all the conditions same as above, but in absence of graphene oxide.The reduction of GO was carried by vapor hydrazine reduction. In brief, the GO or GO-GS sample, kept in a separate beaker, was treated with hydrazine vapor generated by the blowing Ar gas into 10 ml 50% hydrazine hydrate (Sigma Aldrich) for 8 h. During reduction process the samples were heated at 60 °C to stop the condensation of hydrazine on carbon samples. The completion of reduction was judged by the change of brown color of GO to black color of RGO. The reduced hydrazine-treated samples were then termed as N-RGO and N-RGO-GS for the corresponding GO and GO-GS, respectively. At the same time, N-doping was realized on the reduced GO during hydrazine process.The morphology and microstructure of the obtained samples were investigated by scanning electron microscopy (SEM) Hitachi  microscope, Transmission electron microscopy (TEM) EM912 Omega and High resolution-TEM (HR-TEM) JEOL FE-2010 microscope. X-ray photoelectron spectroscopy (XPS) analyses were carried out with an ESCALAB 250 XPS System using a monochromated Al Kα (150 W) source. Raman spectra were recorded with a Renishaw spectrometer using an Ar ion laser (λ = 514.5 nm). The absorption spectra of the samples were recorded by using an Ultraviolet-Visible-Near Infrared Spectrophotometer (CARY 5000) manufactured by Agilent Technology. The specific surface areas of all the samples were measured with ASAP 2020 Physisorption Analyzer  at −196 °C, based on the Brunauer– Emmett–Teller (BET) method from nitrogen adsorption data in the relative pressure range from 0.05 to 0.2. Total pore volumes were determined from the amount of gas adsorbed at the relative pressure of 0.99. The pore size distribution was derived from adsorption branches by the Horvath-kawazoe (HK) report.2) and dried at room temperature overnight, followed by further drying at 80 °C in a vacuum oven for 12 h. The sample loading in all samples are kept constant at 10 mg. The electrochemical performances of the electrodes were tested using a three-electrode configuration in aqueous 6.0 M KOH electrolyte, where prepared carbon sample acts as a working electrode with SCE as a reference and Pt wire as a counter electrode. Cyclic voltammetry (CV), galvanostatic charge–discharge (CD) and electrochemical impedance spectroscopy (EIS) methods were employed for measurement of the electrochemical performance. The CV and CD curves were recorded between −1.0 to 0.0 V at potential scan rates of 10, 50 and 100 mVs−1 and at current densities of 0.1 to 1.0 Ag−1. The EIS measurements were carried out in the frequency range from 10 KHz to 100 mHz with a sinusoidal amplitude of 10 mV. The alternating current (AC) complex impedance was measured based on the following equation Z* = Z′−iZ″. The complex AC capacitance C* = C′−iC″ was calculated from the impedance data, as C′ = Z″/ω|Z|2 and C″ = Z′/ω|Z|2, where ω = 2πf, and f is frequency. The relaxation times τ = 1/fm were calculated from the relaxation frequencies fm, corresponding to the C″ maxima.All the electrochemical measurements were carried out with a Biologic electrochemical workstation (Biologic VSP). N-RGO and N-RGO-GS carbon electrodes were fabricated as follows. The electrode slurry was prepared by mixing 90 wt% active materials and 10 wt% polyvinylidene fluoride (PVdF) binder in a mortar and pestle using a few drops of N-methylpyrrolidinone (NMP) as a solvent. The resulting uniform slurry was then coated onto a nickel foam current collector .


Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing-associated genes via AP1, SP1, MAPK, GSK3α/β and TGF-β signaling pathways, and may promote fast and scarless wound healing in human gingiva. Two connexons from neighboring cells can also dock to form GJs, which provide conduits for direct exchange of small (<1 kDa) signaling molecules between communicating cells [Connexins (C×s) are a family of transmembrane proteins that assemble to form connexons (hemichannels) or gap junctions (GJs). Each connexin protein is composed of four transmembrane-spanning domains, two extracellular loops, and the cytoplasmic domains including N-terminus, C-terminal domain and the cytoplasmic loop. Assembly of six connexin subunits generates one connexon or hemichannel, which functions in autocrine and paracrine signaling by providing a pathway for transfer of signaling molecules, including ATP, NADf small < kDa signng cells . In adding cells –6.Connexins are expressed virtually by all cells in the body, and play crucial role during development and normal tissue function, and contribute to development of various pathologies ,6. In adIn general, fibroblasts in normal skin express connexins, and appear connected to each other by GJs –19. The in vitro [Based on the above studies, C×43 appears a key connexin expressed by skin cells, and it is strongly down regulated in the wound epithelium at the early stages of wound healing. To study its role in more detail, different strategies to further suppress its function or expression in experimental murine skin wounds have been used. For instance, transient blocking of C×43 function at the early stage of wound healing by ACT1, a peptide that binds to the cytoplasmic carboxy-tail of C×43 , or tranin vitro ,25,32,33Interestingly, wound healing in human and pig oral mucosal attached gingiva is faster and results in significantly reduced scar formation as compared to similar skin wounds –37. TherTissue sections from experimental wounds created in palatal attached gingival mucosa in three healthy males (mean age 38 years) that have been previously extensively characterized were used ,42–46. B2. Cells were routinely seeded for experiments when they reached about 95% confluence. Experiments were performed at passages 5 to 10.Four gingival fibroblast lines (GFBLs) were isolated from clinically healthy attached gingiva from healthy human donors, as previously described  S1 Tabl. Cells wGingival tissue donors provided a written informed consent, and procedures were reviewed and approved by the Office of Research Ethics of the University of British Columbia, and complies with the ethical rules for human experimentation that are stated in the 1975 Declaration of Helsinki.2) in their normal growth medium. At day 2 when cultures became confluent, culture medium was replaced with serum-free medium. At day 3, cells were treated with 150 μM of Gap27  [2O) for 24 h before RNA isolation.To block the C×43 function, GFBLs were seeded on 6-well plates  ,49, equa Canada) –50, or c Canada)  or Gap26 Canada) , or corrTo block the expression of C×43, GFBLs were seeded in 6-well plates as described above. After 24 h, siRNA transfection was carried out using Lipofectamine RNAiMax reagent . To this end, 22 μl of Lipofectamine RNAiMax and 150 picomoles of C×43 siRNAs  or control siRNAs  were mixed with 2.4 ml of Opti-MEM medium (Invitrogen), and incubated at room temperature for 5 min. The mixtures of transfection reagent and individual siRNAs were combined and incubated at room temperature for 20 min for complex formation. Then the reagent was diluted with Opti-MEM to yield final siRNA concentration of 30 nM, and added to the cells. After 5 h in cell culture incubator, the transfection reagent was removed, and cells fed with their normal growth medium overnight. Medium was then replaced with serum-free growth medium, and RNA isolation and sample collection for Western blotting was performed after 48 h.http://www.leonxie.com/referencegene.php). Non-transcribed RNA samples were used as a negative control. The PCR reactions were performed in triplicate for each sample. The data was analyzed and is presented based on the comparative Ct method .Real-time PCR analysis was performed according to MIQE guidelines  as we ha2O. Lysates were collected using a rubber policeman, and filtered through a NucleoSpin Filter (Macherey-Nagel) by centrifugation at 5,000g for 10 min.Confluent GFBL cultures were treated as described above. The conditioned medium was then collected and immediately treated with Complete Protease Inhibitor Cocktail . The samples were concentrated (30–40 times) by centrifugation  using Centrifugal Filter Units  for 3 h, and stored at −80°C until use. To collect cell lysates, cells were washed with ice-cold phosphate-buffered saline (PBS), and lysed with a buffer containing 25 mM Tris-HCL (pH 7.6), 100 mM Octyl β̃D-glucopyranoside, 5 mM NaF, 1 mM Na3VO4, , and the Complete Protease Inhibitor Cocktail (Roche Diagnostics), dissolved in HImmunoblotting analysis was conducted as described in detail previously . BrieflyThe activation of signaling pathways by Gap27 treatment was studied in cell lysates obtained as described above. For the experiments, GFBLs were seeded on 6-well plates, treated with Gap27 (150 μM) or equal amount of control peptide for 1, 2, 6, and 24 h, and cell lysates collected, as above. Western blotting was performed with antibodies against total or phosphorylated forms of β-Catenin and GSK3α/β (β-Catenin pathway), SMAD3 (TGF-β pathway), ERK1/2 and p38 (MAPK pathway) . β-TubulTo identify latent and active MMPs, a set of cell/conditioned medium samples was treated with or without p-aminophenylmercury acetate  at 37°C for 4 h to activate latent enzymes  prior toTo determine the role of key signaling pathways in Gap27-induced gene expression, we blocked TGF-β pathway with SB431542 , MEK1/2 with PD184352 , p38 with SB203580 , GSK3α/β with SB415286 , AP1 with curcumin , and SP1 with WP631  in Gap27-treated cells, respectively. To this end, confluent GFBL cultures were pre-incubated with inhibitors at 37°C for 1 h, and then treated with Gap27 (150 μM) with or without the inhibitors in serum-free growth medium for 24 h. All inhibitors were dissolved in DMSO, and control samples were treated with respective amounts of DMSO only. Total RNA was collected for real-time PCR as described above.2O; 1 h) in DMEM at 37°C, media was removed, and a scrape wound was created through the cell layer with a 10 μL pipette tip, and cells incubated as above with 0.5% Lucifer Yellow  in PBS+ (containing 1 mM Ca2+ and Mg2+) for 5 min at 37°C. Cells were rinsed once with PBS+ and then fixed with 4% formaldehyde at room temperature for 20 min. Nuclei were then stained with 300 nM DAPI (Molecular Probes Inc.) in PBS for 5 min. In a set of experiments, cells were transfected with C×43 siRNA-1, siRNA-2 or control siRNA (30 nM) as above before seeding on the coverslips for 24 h, serum-starved for 24 h, and then subjected to the dye transfer experiment as above. Samples were mounted with Immu-Mount solution , examined using the ECLIPSE 80i Microscope , and images captured using NIS-Elements BR software .To assess the GJ function of C×43, dye transfer assays were performed. To this end, GFBL cultures were generated on gelatin-coated glass coverslips in 24-well plates as described previously . Brieflyhttps://www.adobe.com).To assess role of C×43 in cell migration, GFBLs were grown on gelatin-coated glass coverslips, serum-starved, and pretreated with Gap27 or control peptides (150 μM) for 24 h as above. A wound was then created through the cell layer using a 100 μL pipette tip. Cells were then cultured in DMEM with the peptides in a cell culture incubator, and wound closure recorded over time by standardized digital images taken from the samples using a phase contrast microscope  and a digital camera (Nikon Coolpix 995). In a set of experiments, cells were transfected with C×43 siRNA-1 or -2 and control siRNA-1 or -2 (30 nM) separately as above, seeded on gelatin-coated glass coverslips for 24 h, and serum-starved for 6 h before wounding. Experiments were performed in triplicate per treatment group, and images were captured from three to four different areas of the wound on each coverslip. Wound closure rate was determined measuring the area of the open wound at each time point relative to the area of the wound at the time of wounding using the Adobe Photoshop for Mac software  and then serum starved (24 h) as described above, fixed with 4% formaldehyde at room temperature for 20 min, and then permeabilized using 0.5% Triton X-100 in PBS for 4 min. All samples were then blocked with PBS+ containing BSA (10 mg/ml) and glycine (1 mg/ml) at room temperature for 30 min, followed by an incubation with the primary antibody  diluted For immunostaining of human gingival tissue samples, sections were fixed with cold acetone (−20°C) at room temperature for 5 min. Samples were then washed, blocked, and incubated separately with each primary antibody  overnighAll data is presented as mean +/− standard error of the mean (SEM) from repeated experiments. Statistical analysis was performed by using Student’s t-test, p<0.05 was considered statistically significant. Values obtained from the real-time PCR by the comparative Ct-method were Log2 transformed for statistical testing .Previous studies have shown that C×43 is present in human skin, periodontal ligament and gingival fibroblasts, but that skin and periodontal ligament fibroblasts also express C×32, C×40 and C×45 ,17,57. TIn order to assess expression of C×43 during gingival wound healing, we compared localization of C×43 in unwounded human gingiva, and in experimental excisional gingival wounds 3, 7, 14, 28 and 60 days after wounding by immunostaining . Fibroblin vivo [Having established that C×43 is the major connexin expressed by GFBLs, and that abundance of C×43 plaques is strongly reduced during gingival wound healing, we wanted to find out the functional significance of this down regulation. Down regulation of the number of C×43-positive plaques in wound fibroblasts can result in reduced hemichannel and GJ function. Treatment of cells with C×43 mimetic peptides can be used to specifically block these channel functions , which pin vivo . Therefoin vivo ,61. Treain vivo , also inin vivo . Thus, rin vivo . Therefoin vivo ,63, molein vivo  , and studied the expression of the above wound healing-associated genes. Treatment of GFBLs with both C×43 siRNAs consistently resulted in about 80% down regulation of C×43 at both mRNA and protein levels, while expression of the two other connexins expressed by these cells, C×32 and C×45, were not affected (data not shown). C×43 siRNA treatment with both C×43 siRNA-1 and -2 effectively suppressed GJ-mediated dye transfer , but didAs gene expression analysis showed that blocking of C×43 function by Gap27 strongly regulated expression of several genes Tables –4, we fuHaving established that during gingival wound healing, C×43 abundance and/or GJ and hemichannel functions maybe strongly reduced, and that suppressing C×43 function in cultured fibroblasts distinctly regulates expression of a set of wound healing-associated genes, we wanted to find out which intracellular signaling pathways are involved. To this end, we treated GFBLs with Gap27 and assessed phosphorylation changes in TGF-β (SMAD3), MAPK (ERK1/2 and p38), GSK3α/β and β-Catenin pathways that have been previously associated with C×43-mediated signaling ,67–73, aIn order to further assess the role of the above pathways in Gap27-induced gene expression changes, we blocked TGF-β signaling by SB431542, MEK1/2 by PD184352, p38 by SB203580, and GSK3α/β by SB415286 in Gap27-treated cells, and assessed gene expression changes relative to untreated cells by real-time PCR. In addition, we assessed involvement of AP1 and SP1, transcription factors previously linked to C×43 signaling ,79,80, b2) hemichannel plaques [in vivo likely represent C×43 hemichannel and/or GJ plaques on the cell membrane.Gingival fibroblasts that participate in scarless oral wound healing expressed C×43 as the major connexin, which is similar to skin fibroblasts ,21. In o plaques . Connexi plaques . Thus, tin vivo are not clear, but are likely cell type-specific and could include effects of wound healing-related cytokines, growth factors and mechanosignaling [Interestingly, the C×43-positive plaques found in fibroblasts in unwounded tissue were missing from fibroblasts at the wound edge and those migrating into the wound at days 3 and 7 post-wounding. This could be explained by reduced recruitment of C×43 to hemichannel and GJ plaques, or their redistribution, resulting to smaller plaques undetectable by immunostaining, or from down regulation of C×43 expression and/or its increased turnover. The factors that modulate these processes during wound healing ignaling ,84. In aignaling , blockinignaling ,86. Repaignaling . Howeverignaling . Our preignaling ,44,45. Hignaling , suggestignaling , and in ignaling . In addiignaling . Thus, rignaling , 43–46, i.e. it is needed to suppress expression of these genes. Therefore, during gingival wound healing reduced expression/function of C×43 in fibroblasts may allow increased expression of these molecules. This group of molecules includes several MMPs, TIMP-1 and -3, Tenascin-C, TGF-β1 and VEGF-A, which are important modulators of inflammation, cell migration, angiogenesis and ECM deposition [in vivo. The hallmark of scar formation and fibrosis is increased accumulation of ECM and increased cell contractility [in vivo [To study the significance of reduced C×43 function for these later stages of wound healing, we blocked its function in human gingival fibroblasts by C×43 mimetic peptide Gap27 [position , and mayposition ,37. Of nposition ,42, suggactility . Therefoactility , while u[in vivo , were si[in vivo . There i[in vivo ,37,53,93The mechanisms of the mimetic peptide-induced gene expression change in gingival fibroblasts may include blocking of transfer of signaling molecules via hemichannels and/or GJs, peptide-induced changes in C×43 levels, conformation and/or phosphorylation of its cytoplasmic tail that interacts with the signaling molecules ,72,94. TIn order to study the role of the above signaling pathways in regulation of Gap27-induced fibroblast gene expression in more detail, we used pathway-specific pharmacological inhibitors. In addition, we blocked two transcriptional regulators (AP1 and SP1) that associate with C×43 signaling ,79,80. BAs mentioned above, C×43 can also promote TGF-β-induced signaling . InteresTreatment of gingival fibroblasts with Gap27 also induced fast phosphorylation of GSK3α/β, but this did not associate with marked changes in the phosphorylation or levels of its downstream target β-Catenin. Phosphorylation of GSK3α/β renders it inactive removing its inhibitory effect on its targets, which include more than 40 proteins and transcription factors . TherefoInterestingly, Gap27 treatment also significantly increased expression of C×43 at both mRNA and total protein levels, although it did not affect relative proportions of the differently phosphorylated forms of the protein in the Western blots. Increased C×43 mRNA expression was also noted after MFA treatment. These findings are different from previous observations where treatment of human skin fibroblasts with Gap27 in a scratch wound model did not upregulate C×43 levels, but promoted its phosphorylation at S368 . It is pIn order to assess the mechanisms of Gap27-induced C×43 upregulation in gingival fibroblasts we used pharmacological inhibitors to key signaling pathways that associate with C×43. Previous findings have indicated that transcriptional modulator AP1 regulates C×43 expression by various signals . Accordiin vivo warrant further investigation.To summarize, our findings demonstrate that C×43 shows similar spatiotemporal regulation in gingival wound epithelium over time as previously describe for skin. In addition, we showed for the first time that the abundance of C×43-positive plaques is strongly reduced in fibroblasts at the early stages of human gingival wound healing, returning to the level of normal tissue by day 60 post-wounding. Thus, wounding-induced suppression of C×43 in wound fibroblasts leads to disruption of the connexin-mediated intercellular communication network in the connective tissue, resulting to a gene expression change that maybe important for the fast and scarless wound healing outcome in gingiva. Interestingly, blocking of C×43 function by mimetic peptides strongly regulated expression of a number of wound healing and scar formation-associated genes in human gingival fibroblasts. These changes involved p38, MEK1/2-ERK1/2, TGF-β-SMAD3 and GSK3α/β mediated signaling pathways, and AP1 and SP1 transcription factors. Among these pathways, ERK1/2-MEK1/2 appeared to be a key regulator of C×43 mimetic peptide-modulated gene expression. Thus, C×43 mimetic peptides may provide an efficient tool to modulate fibroblast gene expression during wound healing. The exact mechanisms by which the C×43 mimetic peptides cause these effects, and the mechanisms and importance of C×43 down regulation for fast and scarless wound healing outcome in human gingiva S1 FigRepresentative immunostainings of C×43 (red) and vimentin  in unwounded human oral mucosal tissue (attached gingiva) (A–C), and in gingival wounds 3- (D-F), 7- (G-I), 14- (J-L), 28- (M-O) and 60-days (P-Q) post-wounding. (A–C) In unwounded gingiva, abundant C×43 staining was localized in suprabasal epithelial cells. Most intensely stained cells were located in the stratum spinosum, but weak staining was also noted in basal epithelial cells. Inserts in (B) and (C) show higher magnification images of C×43 localization in basal cells at the connective tissue papilla and rete peg areas, respectively. (D–F) At day 3 post-wounding, C×43 was down regulated in migrating epithelial cells (D and E) and fibroblasts at wound edge . (G–I) At day 7 post-wounding, when the wound was completely covered with a new epithelium, 2–3 most basal epithelial cell layers showed C×43 staining in the wound epithelium, while there was only a very weak immunoreactivity for C×43 in the spinous layer (G and H). Very little C×43 immunoreactivity was noted in fibroblasts at the wound edge (I). (J–L) At day 14 post-wounding, C×43 was confined to the 2–3 most basal layers of wound epithelium (J and K). At this stage, immunoreactivity for C×43 was slightly increased at the wound edge connective tissue (L) as compared to 7-day wounds (I). (M–O) At day 28 after wounding, C×43 immunoreactivity was normalized in the epithelium at the wound site, being present mainly in suprabasal cells of the stratum spinosum (M and N). Abundance of C×43-positive plaques in connective tissue cells at the wound edge (O) was increased as compared to earlier time points (I and L). (P–Q) At day 60 after wounding, structure of the epithelium and connective tissue formed at the wound area was closely similar to unwounded tissue. C×43 immunoreactivity was also similar to unwounded tissue in the epithelium at the wound site (P and Q). (R) Negative control staining of C×43 in unwounded epithelium. Data shown represents minimum of three sections stained in parallel samples from two to three individual donors at each time point. Arrowheads  indicate wound edge. E: epithelium; CT: connective tissue; FC: fibrin clot; W Edge: wound edge; WE: wound epithelium; GT: granulation tissue; WCT: wound connective tissue. Nuclear staining (blue) was performed using DAPI. Magnification bars in the inserts in B, C and Q: 50 μm.(TIF)Click here for additional data file.S2 Fig(A and B) Representative images of wound samples double immunostained with anti-C×43 (red) and anti-Clever-1  antibodies. (A) At day 14 post-wounding, very few C×43-positive structures (arrowheads) were noted in some of the macrophages located in the newly made wound connective tissue. (B) At day 28 post-wounding, number of M2 macrophages was strongly reduced compared to day 14, with very little macrophage-associated C×43 immunoreactivity. Magnification bar: 10 μm.(TIF)Click here for additional data file.S3 Fig(A and B) Representative images of the same wound location in parallel day 14 wound sections stained with an antibody against α-SMA (A) and C×43 (B). At day 14 post-wounding, the wound contained numerous α-SMA-positive myofibroblasts. However, very few C×43-positive structures (arrowheads) were noted in cells in the myofibroblast-rich area. Magnification bar: 10 μm.(TIF)Click here for additional data file.S4 FigConfluent fibroblast cultures (GFBL-DC) were cultured in their normal growth medium (DMEM) (A), or treated with control peptide (B) or Gap27 (C) (150 μM), and images acquired 24 h after treatment. Magnification bar: 50 μm.(TIF)Click here for additional data file.S5 Fig(A) Representative images of human gingival fibroblast (GFBL-DC) migration in the presence of Gap27 or control peptide (150 μM) across a scrape wound over time. Lines indicate original wound margins. Magnification bar: 20 μm. (B) Quantification of Gap-27-induced fibroblast migration over time. (C) Representative images of human gingival fibroblast (GFBL-DC) migration in the presence of control siRNA-1 or -2 or C×43 siRNA-1 or -2 (30 nM) across a scrape wound over time. Wounds were completely closed in all groups at 24 h. Lines indicate original wound margins. Magnification bar: 40 μm. (D) Quantification of cell migration in C×43 and control siRNA treated samples over time. Results show pooled data for C×43 siRNA-1 and -2, and control siRNA-1 and -2 treated samples, respectively. Wounds were completely closed in all groups at 24 h. For the experiments, siRNA transfection was performed 30 h before wounding. Wound closure rate was determined measuring the area of the open wound at each time point relative to the area of the same wound at the time of wounding. Results show mean +/− SEM from minimum of triplicate samples. Statistical testing was performed comparing test and control samples at the given time point . Non-treated samples (incubated in DMEM only) did not show difference to control peptide or control siRNA-treated samples, and are not shown.(TIF)Click here for additional data file.S6 FigConfluent fibroblast cultures (GFBL-DC) were treated with Gap26 or control peptide (300 μM), and Gap27 or control peptide (150 μM) for 24 h, and expression of a set of genes was analyzed by real-time PCR. Results show mean mRNA expression relative to control-peptide treated samples from triplicate samples from one experiment. Horizontal line indicates relative mRNA expression for the control-peptide treated samples. EDA-FN: Extra Domain A-Fibronectin; EDB-FN: Extra Domain B-Fibronectin; TN-C: Tenascin-C; α-SMA: α-Smooth Muscle Actin; VEGF-A: Vascular Endothelial Growth Factor-A.(TIF)Click here for additional data file.S7 FigReal-time PCR results from GFBL-DC cultures treated with increasing concentrations of MFA for 24 h relative to vehicle-treated samples are shown. Results represent mean of triplicate samples in one experiment. MFA induced a concentration-dependent increase in expression of MMP-1, MMP-10, Vascular Endothelial Growth Factor-A (VEGF-A), Tenascin-C (TN-C) and C×43, and down regulation of CXCL12 (SDF-1α).(TIF)Click here for additional data file.S8 FigConfluent GFBL-DC cultures transfected with control siRNA-1 (A and B), C×43 siRNA-1 (C) or C×43 siRNA-2 (D) were scrape-loaded with Lucifer Yellow (green), and dye transfer was followed for 5 min. Treatment of cells with C×43 siRNA-1 and -2 reduced markedly dye transfer as compared to control siRNA-1 . Results show representative images from triplicate samples. For the experiments, siRNA transfections were performed 48 h before the experiment. Magnification bars: 50 μm.(TIF)Click here for additional data file.S9 FigConfluent cultures of gingival fibroblasts (GFBL-DC) were treated with Gap27 (150 μM) with or without curcumin (AP1 inhibitor), WP631 (SP1 inhibitor), SB431542 (TGF-β inhibitor), PD184352 (p38 inhibitor), SB203580 (MEK1/2 inhibitor) or SB415286 (GSK3α/β inhibitor) for 24 h, and expression of MMPs and TIMPs (A), ECM proteins and contractility-associated genes (B), TGF-β1 and growth factors (C), and cell-cell junction proteins (D) was analyzed by real-time PCR. Results represent mean mRNA expression relative to non-treated cells from triplicate samples in one experiment. DMSO: Cells treated with the vehicle (DMSO) only; TN-C: Tenascin-C; DCN: Decorin; FMOD: Fibromodulin; α-SMA: α-Smooth Muscle Actin; NMMIIB: Non-Muscle Myosin IIB; VEGF-A: Vascular Endothelial Growth Factor-A.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor (BDNF). In addition to its nervous system functions, TrkB is also expressed in the aortic endothelium. However, the effects of endothelial TrkB signaling on atherosclerosis remained unknown. Immunofluorescence analysis revealed that TrkB expression is downregulated in the endothelium of atherosclerotic lesions from ApoE−/− mice compared with the atheroma-free aorta of WT mice. Endothelial TrkB knockdown led to increased lesion size, lipid deposition and inflammatory responses in the atherosclerotic lesions of the ApoE−/− mice compared with the control mice. Mechanistic studies showed that TrkB activation prevented VE-cadherin shedding by enhancing the interaction between vascular endothelial protein tyrosine phosphatase and VE-cadherin, maintaining VE-cadherin in a dephosphorylated state. Our data demonstrate that TrkB is an endothelial injury-response molecule in atherogenesis. Endothelial BDNF/TrkB signaling reduces VE-cadherin shedding and protects against atherosclerotic lesion development in ApoE−/− mice. Tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor (BDNF). The BDNF/TrkB pathway plays critical roles in the survival, growth, and maintenance of central and peripheral neurons . In addiIn this study, we compared TrkB expression in aortas with and without atherosclerotic lesions in mice and investigated atherosclerotic lesion size, lipid and macrophage components, proinflammatory markers expression in ApoE−/− mice after knocking down endothelial TrkB, seeking to reveal the role and mechanism of endothelial TrkB signaling in the development of atherosclerotic lesions.We performed immunofluorescence staining to compare the expression of TrkB in C57BL/6 mice (controls) and ApoE−/− mice fed with an atherogenic diet for 8 weeks. In agreement with previous results , TrkB exOur previous study revealed that TrkB maintained endothelial barrier integrity . Then thThen the lipid and macrophage deposition in the atherosclerotic lesions of ApoE−/− mice was evaluated. Lipid deposition in the lesion, as demonstrated by the Oil Red O-positive region, was significantly increased in ApoE−/− mice infected with AAV9-shTrkB Figure . The infOur previous study revealed that TrkB promoted VE-cadherin synthesis in HAECs . As expeIt has been reported that an extracellular fragment of VE-cadherin can be cleaved and detected in medium . Then weTyrosine phosphorylation processes of VE-cadherin are required for the VE-cadherin cleavage . Then, wAs a transmembrane binding partner of VE-cadherin, vascular endothelial protein tyrosine phosphatase (VE-PTP) binds directly to VE-cadherin. The binding maintains VE-cadherin in a dephosphorylated state . Then, wIn this study, we investigated the role of endothelial TrkB signals in the development of atherosclerotic lesion in ApoE−/− mice. Our data showed that TrkB expression in the endothelium was downregulated in atherosclerotic lesions. Endothelial TrkB knockdown led to increased lesion size, lipid deposition and inflammatory responses in the atherosclerotic lesions of the ApoE−/− mice compared with the control mice. Finally, TrkB activation prevented the VE-cadherin shedding by maintaining the interaction between VE-PTP and VE-cadherin and reducing tyrosine phosphorylation of VE-cadherin. Our data supported that endothelial TrkB signaling may be a novel therapeutic target for atherosclerosis.In vitro experiments we further demonstrated that the TrkB levels in HAECs were significantly decreased by proinflammatory factors. Our data suggest that TrkB is an endothelial injury-response molecule in atherogenesis and TrkB may participate in the pathologic process of atherosclerosis. Consistently, the decreased BDNF/TrkB levels were also found in the tissues of aged or patients with CAD-related complications. Decreased expression of BDNF in the endothelium was associated with hypertension [TrkB is a high-affinity receptor for BDNF with well-established functions in the nervous system. However, TrkB is also expressed in the aortic endothelium. In this study, we found that TrkB expression in the endothelium to be downregulated in atherosclerotic lesions of ApoE−/− mice compared with the atheroma-free aorta of WT mice. rtension . In humartension . In the rtension . The reaIn this study, we revealed a novel role of endothelial TrkB in cardiovascular system as an atheroprotector. Our data demonstrated that endothelial BDNF-TrkB pathway deficiency led to accelerated development of atherosclerotic lesions in the aortic trees and aortic root of ApoE−/− mice. Similarly, as a growth factor signal, angiopoietin-1 has been reported to protect adult vasculature from leakage . The admIn this study we found that BDNF/TrkB signaling prevented the TNF-α induced-VE-cadherin shedding. The elevated shedding fragment of VE-cadherin has been found to be associated with diabetic retinopathy and coronary atherosclerosis , 24. ThuOur data demonstrate that TrkB expression in the endothelium is downregulated in the atherosclerotic lesions of ApoE−/− mice. In addition to promoting the synthesis of VE-cadherin, endothelial BDNF/TrkB signaling also regulates the shedding of VE-cadherin and protects against atherosclerotic lesion development in ApoE−/− mice. Endothelial TrkB may be used as a novel therapeutic target in atherosclerosis.11 vg/g body weight particles of adeno-associated virus serotype-9 (AAV9) with or without shRNA specifically targeting TrkB via the tail vein [All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health  and were approved by the Ethics Committee of Shandong University. Eight-week-old male ApoE−/− mice received a single systemic administration of 2.1×10Oil Red O staining was used to assess the size of the atherosclerotic lesion as previously described . BrieflyImmunofluorescence staining was used to show the expression of TrkB and VE-cadherin, and macrophage content of the lesions. Briefly, the cryosections of the aortic sinus were fixed with 4% paraformaldehyde. After washing with PBS and blocking with serum, the sections were incubated with anti-TrkB (sc-12), anti-CD31 (Abcam), anti-VE-cadherin , or anti-mouse macrophage  antibodies. Then, the FITC or TRITC-conjugated secondary antibodies were used. Finally, the sections were observed using fluorescence microscopy.Antibodies for anti-TrkB (sc-12), anti-VE-cadherin (C-19), and anti-vascular endothelial protein tyrosine phosphatase (sc-65228) were purchased from Santa Cruz. The preparation of the cell lysates, measurement of the protein concentrations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer, sequential incubation with primary and second antibodies and film development were performed as previously described .ΔΔCt calculation.Total RNA was isolated from the aortas of the mice. First cDNA was synthesized using AMV reverse transcriptase. PCR was performed using the following primers: forward, 5′-AGC AGG GAA ACA TCT ATA ACG-3′ and reverse, 5′-CTT GAA CTT TGG GTT TAC TGG-3′ for mouse VE-cadherin; 5′-AGG CTT CTG GGC CTT ATG TG-3′ and 5′-GTA TTC CTG GCG AGA GAA GCA-3′ for NF-κB; 5′-GTG AAG GGA ATG GGT GTT-3′ and 5′-GGT CAC TGT CCC AGC ATC-3′ for TNF-α; 5′-TTC CAG AAA CCG CTA TGA-3′ and 5′-GGT TGT CAC CAG CAT CAG-3′ for IL-6; 5′-GGC ACC CAG CAG AAG TTG TT-3′ and 5′-CTT GGT AGA GGT GAC TGA GG-3′ for ICAM-1; 5′-TCT CTC AGG AAA TGC CAC CC-3′ and 5′-GTG TGC TGC TAT TGG CTG TG-3′ for VCAM-1; 5′-TCC TCT GGA GAG TGG AGT GC-3′ and 5′-ATG TGA AGC TTT GAC CCA CC-3′ for E-selectin as well as 5′ -TGT CTC CTG CGA CTT CAA CA-3′ and 5′-GGT GGT CCA GGG TTT CTT ACT-3′ for mouse GAPDH. The relative expression of the genes was obtained through the 2Human aortic endothelial cells (HAEC) were purchased from ATCC . The HAECs were cultured in endothelial cell medium. All cells were cultured at 37°C with 5% CO2. All assays were performed in triplicate.P < 0.05 set as the significance level, and they were performed using the SPSS 15.0 software .The animal study data are presented as the mean ± SEM. For comparisons between two groups, Student's t test was employed. All statistical tests were 2-tailed with