Using Rhodamine 6G (R6G) as a probe, SERS spectra were compared on MoS2 N-donuts/graphene nanohybrids substrates. A remarkably high R6G SERS sensitivity up to 2 × 10−12 M has been obtained, which can be attributed to the more robust LSPR effect than in other TMD nanostructures such as nanodiscs as suggested by the finite-difference time-domain simulation. This result demonstrates that non-metallic TMD/graphene nanohybrids substrates can have SERS sensitivity up to one order of magnitude higher than that reported on the plasmonic metal nanostructures/2D materials SERS substrates, providing a promising scheme for high-sensitivity, low-cost applications for biosensing.Nanohybrids of graphene and two-dimensional (2D) layered transition metal dichalcogenides (TMD) nanostructures can provide a promising substrate for extraordinary surface-enhanced Raman spectroscopy (SERS) due to the combined electromagnetic enhancement on TMD nanostructures via localized surface plasmonic resonance (LSPR) and chemical enhancement on graphene. In these nanohybrid SERS substrates, the LSPR on TMD nanostructures is affected by the TMD morphology. Herein, we report the first successful growth of MoS Tancement ,6,7 In amentally ,10. 2D amentally ,12 are ementally  in nanohmentally ,16,17,182 and MoS2) of lateral dimension ~500 nm and height ~3–5 nm grown on the monolayer of graphene (TMD N-discs/graphene), [−12 M on the TMDs N-discs/graphene nanohybrids [−1 on different substrates, the EM enhancement factors could be estimated using graphene only SERS substrate as reference. Specifically, the enhancement factors of 10.3 and 8.7, respectively, are obtained by dividing the intensities of the 613 cm−1 peak on TMDs (WS2) N-discs/graphene and gold nanoparticles (AuNPs) on graphene (or AuNPs/graphene) SERS substrates by that on graphene [While metallic plasmonic nanostructures have been the major focus to induce EM enhancement, nonmetallic ones have recently emerged as a promising alternative to address the drawbacks of the metallic plasmonic nanostructures including strong spectra noise background due to contamination on the metal surface, the deformation and distortion of the probe molecules due to the strong metal-adsorbate interactions, the metal-catalyzed side reactions of the probe molecules and substantial joule dissipation ,10,19,20aphene),  a strongohybrids . It shouohybrids ,28. Usingraphene . While tgraphene ,30.Quantitatively, SERS enhancement relies on many factors such as the size and the shape of plasmonic nanostructures and substrate morphology ,32,33,342 nanodonuts (N-donuts) by controlling the size of the (NH4)2MoS4 precursor nuclei via variation of its concentration in the range of 0.06−0.40 wt%. The hypothesis is that MoS2 N-donuts could be achieved on large-size precursor nuclei due to the nonuniform conversion to MoS2 along the radial direction while MoS2 N-discs form on smaller nuclei when such nonuniformity is negligible. In addition, the solution-based deposition may lead to nonuniform distribution as the solvent is evaporated, which could also lead to donut-shaped structures. With increasing precursor concentrations, the precursor nuclei dimension increases monotonically and MoS2 N-donuts become predominant at precursor concentration of 0.26 wt% or higher. Interestingly, a resonant (at 532 nm excitation) R6G SERS sensitivity ~2 × 10−12 M has been achieved on these MoS2 N-donuts/graphene SERS substrates, in contrast to 5 × 10−12 M for the 0.06 wt% MoS2 where MoS2 N-discs are more prevalent [In this work, we explore the growth of MoSrevalent ,43,44,452 (7 sccm) and CH4 (40 sccm). For graphene transfer on SiO2/Si substrates via a wet transfer procedure [3 solution) for about 4 h, followed by rinsing the PMMA/graphene samples at least five times with deionized water. The PMMA/graphene samples were then placed onto the SiO2/Si and left overnight to dry at room temperature in air. Afterward, the PMMA/graphene/SiO2/Si stack was soaked in acetone four times to remove PMMA. Finally, the samples were rinsed with isopropanol (IPA) and thermally annealed at 400 °C for 30 min in forming gas (Ar (500 sccm)/H2 (300 sccm)) to remove PMMA residues on graphene.Graphene was synthesized on polycrystalline Cu foils  using chemical vapor deposition (CVD) and the details of the growth procedure have been described in detail in our earlier works ,47,48. Brocedure ,47, 3% P2 was grown on graphene using a vapor transport process established in our previous works [2 N-donuts, the (NH4)2MoS4 precursor solution of variable concentrations was prepared by dissolving (NH4)2MoS4 powder in  with different concentrations of 0.06 wt%, 0.10 wt%, 0.20 wt%, 0.26 wt% and 0.40 wt%. It should be noted that continuous layers of MoS2 were achieved at ~0.10 wt% with multiple precursor dips [2 N-donuts could be obtained on graphene with a single dip of the precursor of low (0.06 wt%) to high (0.40 wt%) precursor concentrations. However, at the lower end of precursor concentrations in this range, MoS2 N-discs seemed preferable due to possibly the small precursor nuclei dimension. The nuclei dimension increases with the precursor concentration, MoS2 N-donuts became predominant at the higher end of precursor concentrations. After the graphene/SiO2/Si samples were dipped in the precursor solution, spinning at 3000 rpm for 30 s was followed to achieve a uniform coating of the precursor on graphene. The samples were annealed afterward in a tube furnace at 450 °C for 30 min in sulfur vapor transported by a mixed gas of H2 (10 sccm) and Ar (50 sccm) from the sulfur powder placed upstream from the sample in a quartz tube at about 200 °C.MoSus works ,49. To o2 N-donuts were measured using the Nanoscope software . Specifically, densities were measured using the particle analysis feature, and the diameters and heights were measured by taking cross-sections through at least 20 MoS2 N-donuts for each sample.The sample phase and morphology were characterized using Raman spectroscopy and microscopy , and atomic force microscopy (AFM). For Raman characterization of graphene, 488 nm excitation laser was employed using a 20× microscope objective. The integration time was 3 s and the laser power was ~1–5 mW. For the Raman spectroscopy and maps of TMDs, a 100× microscope objective was used with either 532 or 488 nm laser excitation. For consistency, Raman maps were collected at a minimum of 4 different locations on each sample. AFM images were collected using a Digital Instruments Multimode Nanoscope IIIA AFM system in contact mode using standard silicon nitride cantilevers . Images were collected in at least three locations on the sample surface at multiple scan sizes. The average diameter, height, and density of the MoS−5 M to 10−13 M) was placed on the substrate surface and left to dry for about one hour on a hotplate set at 70 °C. The diameter of the R6G samples on the substrate after it dried is around 4–5 mm. R6G SERS characterization was carried out using laser excitation of 532 nm on the same WiTec alpha 300 confocal Raman system. The laser beam spot was about tens of µm that is further reduced through a 20× microscope objective to allow multiple scan spots near the center of the droplet for consistency. An integration time of 3 s and a low intensity (~1–5 mW) were used to avoid the possible damage of the R6G probe molecules. For an improved signal-to-noise ratio, each presented Raman spectrum is an average of 5–10 spectra gathered at the same spot of the sample. It should be noticed that every Raman spectrum demonstrated in this work is representative because it is based on an average of ~10 or more Raman spectra gathered at different spots arbitrarily chosen on a sample and typically 2–3 samples were tested for reproducibility. To prevent the coffee-ring effect, the tested spots were chosen not too close to the R6G sample’s edge.For the investigation of SERS, R6G was used as a probe molecule. A droplet of R6G  was used. Our simulation model is mostly used to highlight how these two structures support different plasmonic field enhancement factors, defined as the ratios of the field intensities in the presence of the N-donuts and N-discs to those in their absence. Simulations were carried out considering metallic N-donuts and N-discs with different sizes, placed on SiO2 substrates.A finite-difference time-domain simulation (FDTD) study of the characteristic plasmonic effect was carried out to reveal the differences between MoS2 N-donuts/graphene nanohybrids (see Method for details). Controlling the (NH4)2MoS4 precursor concentration in a DMF solution was found critical to obtaining the MoS2 N-donuts. N-donuts nanostructures can be obtained on graphene with precursor concentrations as low as 0.06 wt% while precursor nuclei lateral dimension increases with the precursor concentration. At lower precursor concentrations, MoS2 N-discs seem to be dominant on smaller precursor nuclei  and with 0.26 wt% precursor concentration  (−1 due to the primary in-plane vibration mode and 2D peak at ~2707 cm−1 due to secondary in-plane vibration of zone-boundary phonons [−1 (associated with the defect in graphene8) are clearly observable on these samples. On the pristine graphene, the intensity ratio of 2D to G peak (I2D/IG) is around 1.8 while ID/IG is negligible, indicating that the graphene is a monolayer with high quality. On the MoS2/graphene nanohybrid made with 0.10 wt% precursor concentration, the graphene’s Raman peak intensities are notably enhanced most probably due to the LSPR effect by the MoS2 nanostructures [2/graphene nanohybrid sample made with 0.26 wt% precursor concentration, indicating a stronger LSPR effect. It should be noted that such an enhancement in the Raman signatures of graphene by metallic plasmonic nanostructures has been reported on metal-nanostructures/graphene nanohybrid samples [2/graphene nanohybrids indicates that the LSPR can be effectively generated on nonmetallic TMD nanostructures. 2 taken on the MoS2/graphene nanohybrids made with 0.10 wt% (red) and 0.26 wt% (blue) precursor concentrations, respectively, using Raman excitation at 488 nm. The Raman peaks in both spectra can be indexed to the −1 (due to S and Mo atoms in-plane displacement mode) and −1 (due to the S atoms out of displacement mode) of MoS2 [2 was grown on graphene in the MoS2/graphene nanohybrid samples.ominant) . The gra phonons , and D p samples ,51,52. A of MoS2 , confirm2 Ag1 peak taken on the MoS2/graphene nanohybrid samples made with different precursor solution concentrations of (a) 0.06 wt %, (b) 0.10 wt %, (c) 0.2 wt %, (d) 0.26 wt %, (e) 0.32 wt%, and (f) 0.40 wt%. The Raman maps show that the MoS2 formed is not in a continuous layer, rather in the morphology of NPs of approximately round shape in the selected (NH4)2MoS4 precursor concentration range. Quantitatively, the (NH4)2MoS4 precursor concentration exhibits an effect on both the MoS2 NP density and shape. At the lowest precursor concentration of 0.06 wt%, the round shape of MoS2 NPs grows in two different sizes: smaller size with an average diameter of ~200 nm (majority) and larger size with an average diameter of ~1.0 µm with low density. With increasing precursor concentrations in the range of 0.20–0.26 wt%, the size of the MoS2 NPs changes moderately in the range of 300–600 nm, while the NP density increases. Above the precursor concentration of 0.26 wt%, the lateral dimension of the MoS2 NPs increases considerably and the NP density decreases.2 NPs is shown in 2 NPs have been confirmed in the entire range of the precursor concentration 2MoS4 precursor concentration from ~200 nm at 0.06 wt% precursor concentration to 600 nm at 0.26 wt% concentration. However, the average diameter seems to saturate at higher precursor concentrations.AFM images of 5 µm × 5 µm at selected concentrations of 0.06 wt%, 0.26 wt%, and 0.40 wt% are shown in −5 M) probe molecules on MoS2/graphene nanohybride substrates that were synthesized with different precursor solution concentrations from 0.06 wt% to 0.32 wt%, measured using the same excitation of 532 nm wavelength. Most of the R6G Raman peaks are visible on all spectra. Especially, the spectra include the R6G fundamental peaks at 613 cm−1 allocated to the C–C–C ring in-plane vibration mode, 773 cm−1 assigned to aromatic C–H bending mode, 1190 cm−1 and 1648 cm−1 allocated to C–O–C stretching mode and the C–C stretching mode, respectively [2 N-discs are predominant in this sample. In contrast, the highest SERS enhancement is observed on the spectrum for MoS2/graphene nanohybride made with 0.26 wt% precursor concentration (black) on which MoS2 N-donuts are predominant. Quantitatively, the enhancement factor can be estimated using the ratio of the intensity of the R6G Raman peaks on the MoS2/graphene samples made using different precursor concentrations at 613 cm−1 and 773 cm−1 to the ones on the MoS2 (0.06 wt%)/graphene). 2/graphene sample at 0.26 wt% precursor concentration, suggesting that the SERS enhancement is a compromise of the shape, dimension, and density of MoS2 NPs. Interestingly, MoS2 N-donuts are predominant in MoS2/graphene nanohybrides when the precursor concentration is exceeding ~0.20 wt%. The higher SERS sensitivity in this precursor concentration range, especially the peak SERS sensitivity in MoS2/graphene nanohybride made from 0.26 wt% precursor concentration, suggests that MoS2 N-donuts may support LSPR in a more preferable way than other N-discs or other shapes of MoS2 nanostructures.ectively ,54. Amon−5 M to 2 × 10−12 M were cast and dried on the substrate and then Raman spectra were gathered with a 532 nm laser. The R6G Raman spectra are compared at the R6G concentrations of 5 × 10−5 M to 5 × 10−9 M in −10 M to 2 × 10−12 M in −10 M. With further reduction of the R6G concentration to 2 × 10−12 M, most of the R6G Raman signature peaks are still detectable, particularly the vibrational modes with larger polarizability, such as 613 cm−1, 773 cm−1, 1191 cm−1, and 1648 cm−1. Therefore, the R6G sensitivity of 2 × 10−12 M on the MoS2 N-donuts/graphene nanohybrid is about half an order of magnitude higher than that obtained on MoS2 N-discs/graphene [The R6G molecule droplets of varying concentrations ranging from 5 × 10graphene  and abougraphene .−1 peak enhancement factor  on different SERS substrates of graphene only, MoS2 N-discs/graphene [2 N-discs (0.10 wt%)/graphene [2 N-discs+MoS2 N-discs (0.10 wt%)/graphene [2 N-donuts/graphene (made from 0.26 wt% precursor concentration in this work). The result shows that the best enhancement was obtained on the MoS2 N-donuts/graphene.graphene , AuNPs+Mgraphene , WS2 N-dgraphene  and MoS2−1 and 773 cm−1 as a function of the R6G concentration are depicted in 2 (0.26 wt%)/graphene. The R6G Raman peak intensities at 613 cm2 material studied in this paper. However, since LSPRs are ubiquitous optical signatures of charge carriers, as shown below, our model can explain the impact of the shapes of MoS2 structures studied in this paper on the SERS enhancement. The simulations were done considering a normal planar polarized light reaching an NDs or N-donut placed on a SiO2 substrate  of 500, 360, and 240 nm a. The thThe bi-resonance nature of N-donuts allows them to present a robust field enhancement over a wide range of wavelengths. To see this in In the case of N-discs c, howeve2 N-donuts/graphene nanohybrids SERS substrates. The MoS2 N-donuts growth process consists of two major steps: coating of (NH4)2MoS4 precursor at room temperature in air, followed by vapor transport anneal in sulfur vapor transfer process at 450 °C for 30 min. We have found that the predominant MoS2 N-donuts of the larger lateral dimension of 300–700 nm and height of 5–10 nm can be obtained by varying the precursor concentration to exceeding 0.20 wt% to promote nonuniform MoS2 conversion on larger precursor nuclei. This is in contrast to the formation of predominantly MoS2 N-discs of smaller lateral dimension at lower precursor concentrations down to 0.06 wt%. A SERS study using R6G probe molecules has shown that higher R6G SERS sensitivity has been observed on MoS2 N-donuts/graphene nanohybrid substrates than on the MoS2 N-discs/graphene ones. Specifically, the highest SERS sensitivity up to 2 × 10−12 M has been achieved on the MoS2 N-donuts/graphene nanohybrid SERS substrates made using 0.26 wt% precursor concentration. This is a half order of magnitude higher than 5 × 10−12 M on the MoS2 N-discs/graphene SERS substrates. FDTD simulation indicates that MoS2 N-donuts can better support LSPR than their MoS2 N-discs counterparts. This result illustrates the feasibility of tuning TMD nanostructure morphology for high-performance nonmetallic plasmonic nanohybrids for low-cost, large-scale applications in biosensing and other optoelectronics.In summary, we have developed a layer-by-layer growth process for the fabrication of MoS


The in situ investigation of the dynamic growth process and novel assembly phenomena of graphene on liquid copper (Cu) is of great significance to deeply understand the special behavior of graphene and self‐assembly mechanism. Here, the direct observation of the graphene growth and motion behavior on liquid Cu via in situ imaging is reported. Evidence of graphene movement on liquid Cu is offered and it is demonstrated that the translation and rotation behaviors of graphene are affected by the surface condition of liquid Cu. The self‐assembly process of graphene array is also revealed by capturing the dynamic changes of graphene in real‐time. Further analysis highlights the importance of surface energy of liquid Cu and the interaction between graphene building blocks during the self‐assembling process. The growth parameters are also investigated to flexibly control the assembly configuration of graphene arrays. This work provides an insight into the mechanism of graphene motion and assembly behavior that can be used to guide the controllable manipulation of 2D materials and on‐demand fabrication assembly structures with desired properties. This work develops an in situ investigation system to study the growth and self‐assembly of graphene on liquid copper in real‐time. The translation and rotation behavior of graphene are captured for the first time, which is attributed to the fluidity of liquid copper surface. The self‐assembly mechanism is further revealed, enabling the precise synthesis of various assembly structures. Currently, liquid metal catalysts  with a quasi‐atomically flat surface, show great potential in synthesizing uniform graphene with significantly higher quality and faster growth speed. Meanwhile, the intrinsic fluidity of liquid metal provides a platform for precise manipulation of graphene to achieve complex assembly behaviors.The controllable synthesis of large‐size, defect‐free graphene is a main challenge toward its practical application. We discovered that graphene could assemble spontaneously into a super‐ordered structure on liquid Cu surface. Research on this self‐assembly behavior of graphene is of great significance to scalable and cost‐effective fabrication of integrated devices. However, the current exploration of the growth and self‐assembly mechanism only relies on ex situ experiments and theoretical calculations. The experimental setups of CVD are similar to “black box” in most cases. There are some scientific issues still unclear, such as the dynamic growth and self‐assembly process, and the thermodynamics and kinetics mechanism of self‐assembly behavior on liquid Cu. In situ research enables direct visualization of the dynamic process during the crystal growth and evolution, which is expected to help understand the special behavior of graphene on liquid Cu and provide guidance for precisely synthesizing various assembly structures.In our previous works, we have achieved the controllable synthesis of a series of 2D atomic crystal arrays based on liquid metals.Here, we investigate the growth and motion behavior of graphene on liquid Cu via in situ imaging for the first time. We offer explicit evidence to the movability of graphene on liquid Cu at high temperature. The translational and rotational motion of graphene are captured during the growth and assembly, which are related to the condition of liquid Cu surface. We show the dynamic process of graphene self‐assembly behavior and further reveal the self‐assembly mechanism. We demonstrate that both the minimum surface energy and interaction between graphene are crucial to the formation of aligned graphene arrays via self‐assembling. We further explore the influence of the growth parameters on the growth and self‐assembly of graphene. Our work highlights the unique potential of an in situ technique that enables improved understanding of mechanisms in the CVD process. We believe that our in situ investigation could guide the precise assembly of 2D atomic crystals and promote applications in integrated systems.2Figure The schematic of in situ growth of graphene in a CVD reactor is shown in Figure 4. Based on different emissivity between graphene and Cu, they could be distinguished at high temperature. The typical Rad‐OM image of as‐grown graphene is shown in Figure For studying growth and motion behavior of graphene on liquid Cu, we built an in situ investigation system  and an objective . The quantum efficiency of camera was 72% over 525 to 580 nm and decreased rapidly to less than 5% at 1000 nm (on the website of the company). A 20× long working distance microscopy objective was applied to focus the samples and prevent the thermal radiation damage due to the high temperature.−1. In order to prevent the oxidation of Cu, 200 sccm Ar and 10–30 sccm H2 were introduced into the custom‐made CVD reactor during the whole heating process.The melting process of Cu under high temperature was investigated using the in situ system. After electrochemically polished, the 0.05 mm Cu foil  was put on the W  substrate, which was located in the center of the heating zone. The Cu foil was heated to the melting point and the maximum of the heating rate was less than 30 °C min−1. When the temperature reached 1100 °C, 1–2 sccm CH4 was introduced into the custom‐made CVD reactor for growing graphene. The oxide nanoparticles introduced from the ceramic heater tended to be the nucleation sites, serving as good tracers. After 2–5 min, the CH4 was turned off, and the temperature was decreased to room temperature. The whole process was conducted under 200 sccm Ar and 12 sccm H2.First, the Cu foil was put on the W substrate, which was located in the center of the heating zone. After that, the Cu foil was heated to 1100 °C and the maximum of the heating rate was less than 30 °C minOptical images and videos were taken with a radiation‐mode optical microscopy. Raman spectroscopy was performed with WITec alpha 300R system (488 nm excitation wavelength). The low‐resolution transmission electron microscopy (TEM) images were obtained by a high‐resolution TEM system (JEM‐2100) and the operating voltage was 200 kV. Atomic‐resolution scanning transmission electron microscopy (STEM) image was obtained with JEOL ARM‐200F operated at 80 kV.All the images were conducted with image manipulation software developed by the authors. The image was subtracted after dulling edges and denoising from all the images for background deduction and the minimum value was added at each pixel to avoid the negative value. Then, all the pixels were stretched equally to form the processed image. The contrast and brightness of images were adjusted in image manipulation software. The adjusted gray‐scale images were converted into RGB images. The final images were achieved via RGB color analysis to distinguish the nuclei, graphene, and liquid Cu more clearly.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.Supplemental Video 3Click here for additional data file.Supplemental Video 4Click here for additional data file.Supplemental Video 5Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor prognosis. Patients with IPF represent a heterogeneous population with several described clinical phenotypes. More recently, the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis in IPF patients, with an incidence higher than that in the general population, has drawn attention.A 64-year-old woman previously diagnosed with IPF presented to the emergency department with hemoptysis and hypoxemic respiratory failure.High-resolution chest computed tomography revealed bilateral ground-glass opacities associated with areas of consolidation superimposed on the patient's fibrotic background pattern. Diffuse alveolar hemorrhage was confirmed by the presence of hemorrhagic bronchoalveolar lavage fluid. Hematological and biochemical investigations revealed an inflammatory syndrome, moderate anemia, and rapidly progressive glomerulonephritis. Serological analysis revealed perinuclear antineutrophil cytoplasmic antibody positivity and high levels of antimyeloperoxidase antibodies antibodies. The patient underwent kidney biopsy, which revealed necrotizing glomerulonephritis. Clinical and laboratory findings were diagnostic of microscopic polyangiitis with lung and renal involvement.Cyclophosphamide in combination with methylprednisolone was administered as remission induction therapy. The maintenance therapy consisted of mycophenolate mofetil and prednisone.The patient achieved clinical, radiological, and serological remission within six weeks of treatment.The association between IPF and ANCA-associated vasculitis may represent a distinct clinical phenotype. Autoimmune testing for ANCAs should be considered part of the diagnostic work-up and follow-up of patients with IPF because of the clinical and therapeutic implications of developing vasculitis in an already vulnerable patient. Familial IPF accounts for approximately 4% of all cases.Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial pneumonia of unknown cause that occurs primarily in older adults, is limited to the lungs, and is characterized by the histopathological pattern of usual interstitial pneumonia (UIP). IPF is associated with poor prognosis, with a median life expectancy of 2.5 to 3.5 years after diagnosis.,5Disease progression in an individual patient is difficult to predict, as there is a well-recognized clinical heterogeneity of IPF. Currently described clinical phenotypes with differences in clinical behavior include rapidly progressive IPF, familial IPF, combined pulmonary fibrosis and emphysema, pulmonary hypertension associated with IPF, and acute exacerbation of IPF.Few publications have reported the development of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis in patients with an established IPF diagnosis. ANCA-associated vasculitis is an immune-mediated group of diseases characterized by necrotizing vasculitis, which primarily affects the small blood vessels of the airway and kidneys. The major clinicopathological variants are granulomatosis with polyangiitis ( formerly known as Wegener granulomatosis), eosinophilic granulomatosis with polyangiitis ( formerly known as Churg-Strauss syndrome), and microscopic polyangiitis (MPA). ANCA-associated vasculitis reported in association with IPF is mostly represented by MPA with antimyeloperoxidase antibodies (anti-MPO).In this case report, we present a patient with IPF with subsequent development of systemic vasculitis and review the relevant literature regarding pulmonary fibrosis with ANCA-associated vasculitis. Awareness of this possible association is important, as the development of vasculitis in an already vulnerable IPF patient could represent an event with significant mortality.2A 64-year-old woman presented with progressive breathlessness on exertion and bibasilar inspiratory Velcro crackles on chest auscultation. Chest high-resolution computed tomography (HRCT) revealed changes consistent with the UIP pattern Fig. . PulmonaThe patient was a nonsmoker with no relevant environmental antigenic exposure or drug toxicity. Serological studies for collagen vascular disease  were negative. Bronchoalveolar lavage revealed a normal cytology.Based on clinical background and radiological data, a diagnosis of IPF was established (UIP pattern without evidence of a known cause). The patient was started on antifibrotic therapy with nintedanib. Her IPF remained radiologically stable; however, there was a gradual decline in diffusing capacity of the lung for carbon monoxide to 50% predicted at 6 months.One year after IPF diagnosis, the patient presented to the emergency department with hemoptysis and hypoxemic respiratory failure. On admission, the patient was febrile (38.5°C) and exhibited tachypnea 26 bpm). She was hemodynamically stable with an oxygen saturation of 84% on room air, which increased to 96% with 100% oxygen. Evaluation by chest HRCT revealed new bilateral diffused ground-glass opacities associated with areas of consolidation superimposed on the already known patient's fibrotic background pattern  results were negative.Routine hematology and biochemistry investigations revealed inflammatory syndrome (C-reactive protein = 91.5 mg/L) and moderate normochromic, normocytic anemia (Hb = 9.9 g/dL). Rapidly progressive glomerulonephritis was identified by active urinary sediment on urinalysis, microscopic hematuria, and proteinuria (2746 mg/24 h) in the setting of rising blood urea nitrogen (100.7 mg/dL) and serum creatinine (2.51 mg/dL). Due to the presence of hemoptysis, diffuse alveolar opacities, and low Hb levels, diffuse alveolar hemorrhage was considered and confirmed by hemorrhagic bronchoalveolar lavage fluid.The presence of both diffuse alveolar hemorrhage and rapidly progressive glomerulonephritis suggested pulmonary renal syndrome with a primary differential diagnosis including ANCA-associated vasculitis , Goodpasture syndrome, and systemic lupus erythematosus. Serological analysis confirmed perinuclear antineutrophil cytoplasmic antibody positivity and high levels of anti-MPO antibodies (77.2 U/mL). A kidney biopsy revealed necrotizing glomerulonephritis. Clinical and laboratory findings were suggestive of microscopic polyangiitis. the disease severity was graded as generalized active. The patient was administered cyclophosphamide in combination with high-dose glucocorticoids for induction and maintenance therapy. The full therapy consisted of 6 intravenous pulsed cyclophosphamide  and 1 g of intravenous methylprednisolone for 3 consecutive days followed by 0.5 mg/kg/d oral prednisone with subsequent slow tapering to 0.25 mg/kg/d at 12 weeks. Antifibrotic treatment with nintedanib was continued. No treatment-related adverse events were observed.According to the European Vasculitis Study Group classification, The patient remained in remission 6 months after the diagnosis of vasculitis.The patient achieved clinical and serological remission within 6 weeks of diagnosis with renal function recovery, resolution of ground-glass opacities on chest HRCT, and normalization of anti-MPO antibody levels. Maintenance therapy consisted of mycophenolate mofetil (2 g/d) and prednisone (0.25 mg/kg/d). Mycophenolate mofetil was preferred in our patient with IPF, patient considering its potential benefit on lung function through its antiproliferative and antifibrotic effects.3–12 The most frequently detected ANCAs are anti-MPO and much less often antiproteinase antibodies , antielastase, antilactoferrin, or nonspecific ANCA positivity. There are no differences in clinical background, laboratory results, or pulmonary function tests between the ANCA-negative and ANCA-positive IPF patients. One study of IPF patients from North American cohorts found that MPO positivity predominated in women, whereas all PR3-positive individuals were male. Studies from Japanese populations have found equal sex distributions.,11,13Evidence indicates that the prevalence of ANCA positivity in patients with IPF is higher than that in the general population. At the time of IPF diagnosis or during follow-up, up to 20% of patients present with ANCA positivity, without clinical manifestations of systemic vasculitis.,13 These radiological findings translate into predominant lung inflammation, as lung biopsies of patients with ANCA positivity revealed more frequent interstitial inflammation, plasma cell infiltration, lymphoid follicles with germinal centers, and cellular bronchiolitis. ANCA-positive IPF patients may present more neutrophils in bronchoalveolar lavage fluid than patients without ANCA.Chest HRCT studies in ANCA-positive IPF subjects showed a significantly higher prevalence of ground-glass opacities superimposed on the UIP background pattern (increased attenuation around honeycombing).In our patient, ground-glass opacity admixed with reticular abnormality and traction bronchiectasis were present on chest HRCT before the development of clinical vasculitis. This finding was considered part of the fibrotic process. The limitation of the present clinical case is the lack of ANCA evaluation at the baseline diagnosis of IPF and thus the impossibility of correlating the baseline radiological anomalies with a possible ANCA positivity.–12 Significantly few studies have reported the development of granulomatosis with polyangiitis in antiproteinase antibodies positive IPF patients. The most frequently reported clinical manifestations of MPA in the IPF population are rapidly progressive glomerulonephritis, mononeuritis multiplex, diffuse alveolar hemorrhage, and gastrointestinal involvement.,12,15A significant proportion of ANCA-positive IPF patients develop vasculitis. Up to 26% of anti-MPA-positive patients develop MPA with anti-MPO antibodies.,12 However, chronic use of corticosteroids is no longer recommended in IPF, as randomized trials have shown an increased risk of hospitalization and death associated with immunosuppressant therapy. There was no significant difference in survival between ANCA-positive and ANCA-negative IPF patients or between ANCA-positive patients treated or not treated with corticosteroids.,12 Thus, the available data do not support the use of corticotherapy to prevent MPA development in ANCA-positive IPF patients, as benefits outweigh the risks.Several studies have reported a lower incidence of MPA in patients with ANCA-positive IPF patients treated with corticosteroids.The treatment of ANCA-associated vasculitis in patients with IPF follows international guidelines for vasculitis. Combination therapy with corticosteroids and cyclophosphamide is the mainstay of remission induction treatment for severe MPA. One study that compared the prognosis of the UIP pattern in MPO-ANCA nephritis patients with the prognosis of IPF patients found no significant difference in the median survival time.The pathophysiological mechanisms underlying the association between ANCA vasculitis and pulmonary fibrosis remain poorly understood. It remains unclear whether this association represents 2 distinct diseases with potential pathophysiological links or manifestations of the same disorder. IPF inducing ANCA-vasculitis is suggested by the higher age at presentation, similar to IPF alone, and already established pulmonary fibrosis preceding full vasculitis syndrome in the majority of patients. UIP is the most frequent radiological and histological pattern in ANCA-positive pulmonary fibrosis, and appears to be the same as that in IPF. Conversely, ANCA vasculitis can lead to pulmonary fibrosis through recurrent occult alveolar hemorrhage or direct fibrogenicity of anti-MPO antibodies.4IPF with ANCA positivity could represent a distinct phenotype characterized by ANCA-positive conversion (mostly anti-MPO), the presence of ground-glass attenuation around honeycombing on chest HRCT, and possible subsequent development of clinical vasculitis in an IPF patient. Autoimmune testing for ANCAs should be considered part of the diagnostic work-up and follow-up of IPF because of the clinical and therapeutic implications of developing vasculitis.The authors thank the patient for granting permission to publish this manuscript.Conceptualization: Ovidiu Fira-Mladinescu, Diana Manolescu.Investigation: Daniel Traila, Monica Steluta Marc.Resources: Monica Steluta Marc, Camelia Pescaru.Supervision: Ovidiu Fira-Mladinescu.Writing – original draft: Daniel Traila.Writing – review & editing: Ovidiu Fira-Mladinescu, Diana Manolescu.


Poly Adenylate Binding Protein Interacting protein 1 (PAIP1) plays a critical role in translation initiation and is associated with the several cancer types. However, its function and clinical significance have not yet been described in oral squamous cell carcinoma (OSCC) and its associated features like lymph node metastasis (LNM). Here, we used the data available from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) to analyze PAIP1 expression in oral cancer. The publicly available data suggests that PAIP1 mRNA and protein levels were increased in OSCC. The high PAIP1 expression was more evident in samples with advanced stage, LNM, and worse pattern of invasion. Moreover, the in vitro experiments revealed that PAIP1 knockdown attenuated colony forming, the aggressiveness of OSCC cell lines, decreasing MMP9 activity and SRC phosphorylation. Importantly, we found a correlation between PAIP1 and pSRC through the analysis of the IHC scores and CPTAC data in patient samples. Our findings suggest that PAIP1 could be an independent prognostic factor in OSCC with LNM and a suitable therapeutic target to improve OSCC patient outcomes. Like most epithelial cancers, OSCC is a common cancer type of the mucosal linings of the oral cavity. LNM is one of the deterministic prognostic indicators in OSCC2. Despite the multimodal approach, including surgery or radiotherapy as treatment approaches in patients with OSCC, the low LNM-associated survival rate of less than 50% remains a major concern4. Thus, there is crucial to identify novel LNM-related biomarkers to improve the clinical outcomes in patients with OSCC.Tumor metastasis is critical for tumor progression, depending on motility and invasion, cancer cell plasticity, the modulation of the microenvironment, and cancer cell colonization9. PAIP1 displays a PAM1 and PAM2 domain, of which PAM1 interacts with the RRM 1and 2 domains of the Poly Adenylate Binding Protein (PABP) and PAM2 with the C-terminal domain of PABP5. Its interaction with eIF3 is regulated via the S6 kinase10. These interactions help the circularization of the RNA and translation initiation8.The coding gene of PAIP1is located at 5p12 chromosomal location, encoding a protein of 479 amino acids sharing homology with the central domain of human eukaryotic initiation factor 4G (eIF4G), consequently interacting with the eIF4A and eIF3 complexes to facilitate translation initiation11. Similarly, it reportedly regulates different aspects of oncogenesis, such as cellular proliferation, cell death, and metastasis in several cancers15. Although these findings suggest that PAIP1 plays a part in tumor progression and metastasis as a potential prognostic factor, the correlation between PAIP1 expression and OSCC metastasis has not yet been described.Since its identification by Sonenberg et al. in 1998, it has been described to be frequently upregulated in several types of cancer. In breast cancer, the high PAIP1 expression was related to late clinical stage, high histologic grade, and a low survival rateIn this study, we tried to examine whether PAIP1 is frequently overexpressed in human OSCC tissues and cell lines and to investigate the correlation between PAIP1 expression and the clinicopathological parameters of patients with OSCC, just as well as to evaluate the prognostic value of PAIP. Furthermore, to explore the function of PAIP1 and its underlying molecular mechanisms, we observed the migration and invasion in PAIP1-silenced OSCC cell lines.in silico PAIP1 expression analyses. In public database, PAIP1 was frequently amplified and overexpressed in a variety of cancers including head and neck squamous cell carcinoma (HNSCC) . To further evaluate the PAIP1 expression in HNSCC, we assessed differentially expressed genes in 45 healthy and 45 cancer samples from the GSE dataset 30784 and found different, significantly upregulated PAIP1 transcripts  Fig. . We alsopts Fig. . Comparepts Fig. . Moreovepts Fig. . Consistnes Fig. . Based ones Fig. . These rin silico data . In a recent comparative study, the worst pattern of invasion (WPOI) was reportedly superior to histological grade as a prognostic indicator of OSCC16. To investigate the relevance of PAIP1 expression on WPOI, we categorized it into 5 different patterns and classified it into two categories: cohesive and non-cohesive, following their grading schemes  and ITF (invasive tumor front). The IHC scores of the PAIP1 staining in ITM or ITF are shown as a heat map. The ITF exhibited significantly higher IHC scores than ITM  using siRNA interference Fig. . First, P- and R-values of 0.000 1 and 0.50, respectively). We performed a multivariable analysis to evaluate the correlation between PAIP1, pSRC (Tyr 419), WPOI, and nodal metastasis, observing a positive correlation between these variables . These data highlighted that SRC phosphorylation is involved in the poor OSCC prognosis with a high PAIP1 expression. A summary of the working model by which PAIP1 has metastatic potential and prognostic significance in OSCC is illustrated in Fig. To examine the involvement of PAIP1 in SRC phosphorylation, closely related to tumor invasion and metastasis, we performed western blot in PAIP1 knockdown cells. The SRC (Tyr416) activation was strongly attenuated by PAIP1 inhibition in both cell lines Fig. . We thenles Fig. . Finally6. Amplification of short arm of chromosome 5 is frequently seen in cancers18. This led us to hypothesize that PAIP1 might undergo amplification in head and neck cancer. Concurrent with our hypothesis, we found that the genetic alterations seen in head and neck cancers were mostly amplifications with mutations and fusions and a strong correlation between PAIP1 copy numbers and mRNA levels  when compared to inner regions (ITM)  show molecular heterogeneity when compared to the preceding zones of the tumor, which can better predict the tumor outcomesTM) Fig. . PAIP1 eTM) Fig. 24,25. ThLNM Fig. . FurtherLNM Fig. . HoweverLNM Fig.  and S4c.15. In particular, Guan et al. reported that PAIP1 knockdown in pancreatic cancer cells markedly decreased in the expression of MMP2, which affects the invasion and migratory process and MMP2 in OSCC has also been described as a potential biomarker for metastasis33, whereas we found no change in the MMP2 levels, rather MMP-9 activity downregulation in the PAIP1 knockdown of OSCC cells 15. Against to our expectations, these findings indicate that EMT-related molecules regulation of PAIP1 was unlikely to be pertinent in our model. If the migratory and invasive abilities of OSCC cells are blocked by PAIP1 knockdown, it may still occur by an alternative mechanism.Although the recently growing pieces of evidence suggest that high PAIP expression contributes to the aggressive behavior of various tumors, its role has never been determined in OSCCls Figs.  and S61536. Wang et al. reported that PAIP regulated AKT/GSK3β pathways for migration in lung cancer14. Although we did not find any significant changes including the AKT signaling pathway (data not shown), we for the first time found that PAIP1 was associated with activation of SRC by affecting phosphorylation of tyrosine residue 416/419  were used to extract expression data and identify PAIP1 expression and its relationship with the clinicopathological features in HNSCC.Three data repositories . Reference for the codes developed was accessed from, https://labs.cd2h.org/gitforager/repository/repository.jsp?id=77901462. The results shown here are partially based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.HNSCC dataset was used to create a custom cohort, which was accessed via https://pdc.cancer.gov/pdc/ including the base of the tongue NOS (Not Otherwise Specified), cheek mucosa, lip NOS, gum NOS, tonsil NOS, the floor of the mouth NOS, head, face, or neck NOS, just as well as the overlapping lesions of the lips, oral cavity, and pharynx. The log values for reporter ion intensity for unshared PAIP1 peptides were extracted after the manual downloading of the files CPTAC3_Head_and_Neck_Carcinoma_Proteome.tmt11.tsv and CPTAC3_Head_and_Neck_Carcinoma_Phosphoproteome.phosphopeptide.tmt11.tsv. The obtained values were used to compare the physiological and cancer-affected expression levels44.We analyzed the proteomic PAIP levels of a custom cohort, accessed from 45 and staging of tumors was based according to the TNM system recommended by the American Joint Committee on Cancer46. We performed a histologic risk assessment to record the worst invasion patterns (WPOI)27. The clinicopathological and histological features of the patients with OSCC are summarized in Supplementary Tables The specimens analyzed were retrieved from 58 OSCC patients who were surgically treated at the Seoul National University Dental Hospital at Department of Oral and Maxillofacial Surgery in 2009. The clinical data were collected from the patient medical records which included age, sex, recurrence, and survival of the cases. The tumor grading was based on the World Health Organization Classification of TumorsFour μm serial sections were obtained from 58 and 36 formalin-fixed and paraffin-embedded OSCC and adjacent healthy oral mucosal tissue specimens, respectively. The specimens were serially sectioned for each case to detect PAIP1 and pSRC (Tyr419). One section per case was immunohistochemically stained using a mouse monoclonal anti-PAIP1 antibody  after heat-induced antigen retrieval in a microwave oven for 10 min in citrate buffer (pH 6.0) incubated overnight at 4 °C, followed by detection using Dako real detect HRP-conjugated system (Dako). The other section was immunohistochemically stained after heat-induced antigen retrieval in a microwave oven for 8 min in citrate buffer (pH 6.0) using anti-pSRC (Tyr419)  incubated for 1 h at room temperature, followed by detection using Dako real detect HRP-conjugated system. Slides incubated without primary antibodies were used as a negative control.21 along with the total tumor score. The percentage of positive cells was scored as follows: Score 0, less than 5% positively stained cells; Score 1, 5%–25% positively stained cells; Score 2, 26%–50% positively stained cells; and Score 3, 51%–100% positively stained cells. The intensity was also scored for positive cells as follows: Score 0, negative; Score 1, weak (light brown); Score 2, moderate (brown); and Score 3, strong (dark brown). Each intensity score was added to the percentage score to produce a combined score. The final IHC score varies between 0 and 6. IHC scores between 0 and 2, less than 4, and equal to or greater than 4 were classified as negative, low, and high, respectively  assessed immunohistochemical staining semi-quantitatively, individually following blinding. In the case of disagreement, the scores were reached to a consensus following discussion. Tumor cells that demonstrated cytoplasmic staining were considered positive. The tumor was demarcated into two different zones: inner tumor mass and invasive tumor front (most advanced layers and detached tumor cells). Both zones were scored independentlyely Fig. . For sta2 until reaching a confluency of 70%–80%.Eight OSCC cell lines were screened along with a positive and negative control for PAIP1 protein and mRNA expression. The Hokkaido University kindly provided the HSC2, HSC3, HSC4, Ca9.22, HO1-N1, and HO1-U1 cells and the Dankook University kindly provided the HN22 cells. The SCC-9 was acquired from the Korean Cell Line Bank and MDA-MB-231 cells were purchased from the American Tissue Culture Collection. The Yonsei University kindly provided the iHOK cells. The MDA-MB-231 and iHOK were used respectively as positive and negative controls. The cells were routinely maintained in DMEM-F12 or RPMI 1640 media, supplemented with 10% FBS and penicillin/streptomycin and KSF medium (for iHOK cells) supplemented with BPE/EGFR at 37 °C, 5% CO−∆∆CT method47. We used the qPCR primers as follows: PAIP1 forward, 5′-TTT GGA AGA TGC TTG GAA GG-3′, PAIP1 reverse, 5′-TGA AGT TGC ATG GAC TCT GC-3′; GAPDH forward, 5′-GTGGTCTCCTCTGACTTCAAC-3′, GAPDH reverse, 5′-CCTGTTGCTGTAGCCAAATTC-3′.1 microgram of RNA was used to reverse transcribe using the AMPIGENE cDNA Synthesis Kit  according to the manufacturer’s protocol and then, the resultant cDNA was subjected to PCR using the AMPIGENE qPCR Green Mix Hi-Rox . The real-time PCR was performed using the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems) and the amplification parameters for all genes were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Fold amplification of each gene was normalized to the amount of GAPDH and calculated using the 2Protein lysates were prepared using RIPA buffer supplanted with a protease inhibitor cocktail. The concentration of protein was determined by the DC Protein Assay Kit. The equal amounts of protein samples were separated on SDS polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. This membrane was blocked with a blocking solution, TBST, supplemented with 5% Difco Skim Milk (BD bioscience) for 1 h, the membrane was then incubated with PAIP1-specific antibody (Santa Cruz), anti-GAPDH antibody (Abcam), anti-α-Tubulin (Santa Cruz), anti-SRC , anti-pSRC (Tyr416) , or anti-MMP9  in TBST. Following incubation with HRP-conjugated secondary antibodies, proteins were finally detected using the SuperSignal West Pico Chemiluminescent Substrate and captured using ImageQuant LAS 500 .PAIP1 were provided by Bioneer. The PAIP1 siRNA sequences were as follows: human PAIP1 siRNA (#10605-1): sense 5′-CCAGGUGGUUGUAGCUCCU-3′, antisense 5′-AGGAGCUACAACCACCUGG-3′. The cells were seeded onto 60 mm dishes. The cells were cultured along with 25 nmol·L−1 of sicontrol or PAIP1 siRNA using Lipofectamine 2000 reagent according to the manufacturer’s instructions.Small-interfering RNAs (siRNA) targeting PAIP1 siRNA-treated cells were seeded onto six-well culture dishes (2000 cells per well) and incubated. The medium was changed every 3 days for a week. The colonies (clusters of 20 or more cells) were then stained with 1% crystal violet and taken under an inverted microscope, and the number of colonies was calculated using the Image J software.4 cells per well) were grown in the upper chamber of transwell inserts in 500 μL of serum-free DMEM/F12 medium. To assess cell migration, 8 µm Transwell membranes of chamber (Corning) were precoated on the lower surface with Collagen Type I to yield a concentration of 0.5 mg·mL−1. For the invasion assays, the transwell inserts were precoated with growth factor reduced Matrigel (1:15 dilution). A total of 700 μL of DMEM-F12 with 10% FBS was inoculated in the lower chamber and then, the assay plates were incubated at 37 °C in 5% CO2 for 24 h. The upper chamber was then wiped off with a cotton swab, following this hematoxylin and eosin were used to stain the chamber. The cells were counted under a light microscope . Finally, 10 unique microscopic fields at ×100 magnification from each chamber were randomly chosen for cellular counting.The siRNA-treated cells  at 37 °C for 72 h. The gel was then stained with Coomassie Brilliant Blue R250 (Bio-Rad Laboratories). Thereafter, de-staining of the gel was performed using a solution of 40% methanol and 10% acetic acid, until the MMP-9-degraded part of the membrane became clear. Evaluation of the density of the clear bands was done using the ImageJ software.The siRNA-treated groups were sustained in a serum-free medium for 72 h. To collect samples of the MMP protein extract, the conditioned medium was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) at 4500 g and 4 °C for 80 min. To assess the MMP-9 activities, 10 and 30 µg protein samples were applied to an acrylamide gel, 8% which was prepared with 1.5 mg·mLP < 0.05 was considered statistically significant.Statistical tests were conducted using the SPSS ver. 25.0  and GraphPad Prism 8 software. All results were expressed as the means ± standard deviation (SD). Multivariable correlations between the immunohistochemical staining and clinicopathological parameters of the tumors were performed via multivariable Pearson correlation, Student’s t-test, the Chi-square test, and Fisher’s exact test. The relative risks were calculated for different variables for high PAIP1 expressions. The statistical significance of the in vitro experimental data was analyzed using the two-tailed Student’s t-test (for comparisons of two treatment groups) or one-way ANOVA with Tukey’s post hoc test (for comparisons of three or more groups). A Supplementary Figure LegendsFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Supplementary Tables


Interstitial lung diseases (ILDs) are a large and diverse group of rare and chronic respiratory disorders, with idiopathic pulmonary fibrosis (IPF) being the most common and best-studied member. Increasing interest in fibrosis as a therapeutic target and the appreciation that fibrotic mechanisms may be a treatable target of IPF prompted the development and subsequent approval of the antifibrotics, pirfenidone and nintedanib. The management of ILDs has changed considerably following an understanding that IPF and some ILDs share similar disease behavior of progressive fibrosis, termed “progressive fibrosing phenotype”. Indeed, antifibrotic treatment has shown to be beneficial in ILDs characterized by the progressive fibrosing phenotype. This narrative review summarizes current knowledge in the field of progressive fibrosing ILDs. Here, we discuss the clinical characteristics and pathogenesis of lung fibrosis and highlight relevant literature concerning the mechanisms underlying progressive fibrosing ILDs. We also summarize current diagnostic approaches and the available treatments of progressive fibrosing ILDs and address the optimization of treating progressive fibrosing ILDs with antifibrotics in clinical practice. Fibrosis refers to an exaggerated accumulation of extracellular matrix proteins in response to inflammation and injury ,2. This ILDs form a large and heterogeneous group of rare and chronic respiratory disorders that present with damage to the lung parenchyma, usually caused by a combination of inflammation and fibrosis . IPF is ILDs have traditionally been treated with off-label corticosteroids and/or immunosuppressive therapies, with variable effectiveness depending on the disease. In particular, the PANTHER trial, showing that the combination of azathioprine, prednisone and N-acetylcysteine worsened the outcomes of IPF patients, provided compelling evidence that inflammatory and immune responses may not be the optimal target . The devIn recent years, the similar disease behavior shared by IPF and progressive fibrosing ILDs has resulted in the hypothesis that antifibrotic treatment may be beneficial in other ILDs characterized by the progressive phenotype . This hyThe results of these trials showed that the antifibrotic, nintedanib, significantly reduced the decline of lung function compared with placebo and to a similar extent to that observed in patients with IPF ,13, resuWhile the availability of targeted drugs is no doubt a major advance in the management of progressive fibrosing ILDs, several practical issues remain challenging, including the diagnosis and classification of these conditions; their variable clinical course; the lack of validated biomarkers for the prediction of disease progression; and the lack of a clear definition of a progressive fibrosing phenotype. The aim of this narrative review is to summarize current knowledge in the field of progressive fibrosing ILDs and to suggest methods for improving the management of these rare conditions with the new drugs. To this purpose, we will first discuss the most relevant literature concerning the mechanisms underlying progressive fibrosing ILDs; we will then discuss current diagnostic approaches and the available treatments. Finally, we will address the optimization of the treatment of progressive fibrosing ILDs with antifibrotics in clinical practice.IPF, the prototype of fibrosing ILDs, is defined in a joined guideline by the American Thoracic Society (ATS), the European Respiratory Society (ERS), the Japanese Respiratory Society (JRS) and the Latin American Thoracic Association (LATS) as a “specific form of chronic, progressive fibrosing interstitial pneumonia of unknown cause, occurring primarily in older adults, and limited to the lungs.” . It is aA multidisciplinary approach involving pulmonologists, radiologists and pathologists improves the accuracy of diagnosis ,21. The Although the etiology of IPF is unknown by definition, several risk factors have been identified, including aging, cigarette smoking, environmental exposures, viral infections and chronic tissue injury  ,24. GeneGenome-wide association studies have identified single-nucleotide polymorphisms (SNPs) in patients with IPF . In partThe 2018 ATS/ERS/JRS/ALAT guideline for IPF diagnosis lists genetic markers among the topics requiring further research and points out that, although genetic variants contribute to the risk of developing IPF, their clinical relevance needs to be demonstrated in future studies .Similar to other chronic diseases resulting in organ failure, IPF results from the combination of genetic susceptibility and environmental risk factors ,29. DespThe injury of alveolar epithelial cells is followed by a cascade of repair processes that involve an inflammatory response with the accumulation of lymphocytes and macrophages at the injured site and release of profibrotic mediators ,24. FibrThe UIP pattern has always been indicative of a diffuse fibrosing pathology with a progressive trend regardless of the precise diagnosis ,21,22,32A proportion of ILDs can have a disease course overlapping with that of IPF, with similar high morbidity and mortality . A recenIn addition to IPF, the ILDs most likely to develop a progressive fibrosing phenotype include idiopathic non-specific interstitial pneumonia; unclassifiable idiopathic interstitial pneumonias; rheumatoid arthritis-associated ILD; systemic sclerosis-associated ILD; hypersensitivity pneumonitis; sarcoidosis; and ILDs related to other occupational exposures  . These dThe recognition that IPF and certain ILDs can share a behavior of progressive fibrosis—termed “progressive fibrosing phenotype”—despite distinct initial disease triggers has considerably changed the management of ILDs . GroupinAn analysis of the INBUILD data showed that there are well-established parameters  that can identify the trend in progression. Specifically, the inclusion criteria of the INBUILD trial were shown to be effective in identifying patients with fibrosing ILDs with a progressive phenotype . Importantly, rapidly non-IPF progressive fibrosing ILD was described in three patients who were younger than the average patient with IPF, had received a non-IPF multidisciplinary team diagnosis and had a non-UIP pattern on HRCT . This caThe clinical practice-oriented definitions of the progressive fibrosing phenotype recently suggested in a position paper from the Erice ILD working group are reported in We have always been accustomed to try to exclude, with all rational efforts and according to good clinical practice, the secondary nature of the progressive disease of ILD. This is because, with our increasing knowledge on the pathogenesis of IPF, the evidence supported the antifibrotic treatment of IPF (nintedanib and pirfenidone) as the only effective treatment capable of improving outcomes. There is no evidence to support antifibrotics in the setting of a non-definitive diagnosis of IPF, despite it being clear that the fibrosis is the same as IPF, and the standard of care, therefore, includes steroids, immunosuppressants and cytotoxic agents.With the evidence to date on nintedanib (SENSCIS and INBUILD), the scenario has changed. A diagnosis based on clinical behavior  is sufficient for using an antifibrotic with similar success compared to that of IPF. It could be argued that the precise diagnosis is the first aim of the clinical procedure in order to prescribe suitable therapy. Indeed, the question arises as to whether our judgment of suitable therapy would remain the same if the results of SENSCIS and INBUILD had been available earlier. HRCT is important for the diagnostic evaluation of fibrosing ILDs, as reviewed by Torres and colleagues . MoreoveA 2018 review on the role of imaging in progressive fibrosing ILDs not only identified HRCT as central to the diagnosis in ILDs but also pointed out the need to identify new methods for image analysis, which quantify the extent and progression of fibrous pathologies using a machine learning-based approach . The linHence, an optimal approach to disease staging and outcome prognosis in fibrosing ILDs with a progressive phenotype may be achieved by combining semi-quantitative imaging techniques, based largely on visual analysis, with quantitative imaging coupled to machine learning .Lung biopsy, alongside the development of less invasive procedures, is also useful in the diagnosis and assessment of the progressive fibrotic phenotype, as recently reviewed by Ravaglia and colleagues .Several serum biomarkers have been tested for their ability to predict disease progression, but no molecule has been validated as a prognostic marker for use in clinical practice so far . As for Current understanding is limited regarding the pathogenesis of progressive fibrosing ILDs. Some ILDs are primarily fibrotic, with fibroblast dysfunction resulting in fibroblast proliferation and fibrosis, as discussed above for IPF. Other ILDs are predominantly inflammatory disorders but can shift, via mechanisms that are currently unknown, to a fibrotic pathway under certain conditions. Although the initial pathogenetic mechanisms are distinct between these two groups of ILDs, the processes resulting in self-sustaining fibrosis and organ damage are likely to be common ,50. In the latest update of the ATS/ERS/JRS/ALAT guideline for IPF treatment published in 2015, nintedanib and pirfenidone, along with antacid therapy, are the only drugs to be recommended for the treatment of IPF  . NintedaCurrently, there are no treatment guidelines issued by a scientific society to guide clinicians in the treatment of progressive fibrosing ILDs other than IPF. Treatment of these conditions usually involves corticosteroids or immunosuppressants, including azathioprine, mycophenolate mofetil and cyclophosphamide . Howeverp < 0.001). The adverse event profile associated with nintedanib was acceptable and similar to the one observed in IPF. According to a subgroup analysis of the INBUILD trial by ILD diagnoses, nintedanib reduced the rate of disease progression equally across the five ILD subgroups, regardless of the underlying diagnosis [Given the general lack of evidence supporting immunomodulatory treatments for progressive fibrosing ILDs other than IPF, as well as the lack of efficacy of these treatments in many patients, the results of the trials investigating antifibrotic drugs in patients affected by these conditions ,13,15 ariagnosis . Safety Pirfenidone has so far been investigated for the treatment of progressive fibrosing unclassifiable ILD in a phase 2 trial involving 253 patients . The prip = 0.013) [Our understanding of the potential of antifibrotics for the treatment of progressive fibrosing ILDs is likely to improve soon, as a number of clinical trials with nintedanib and pirfenidone are ongoing . Other a= 0.013) . Pamrevl= 0.013) ,56. A phKey issues for improving the treatment of IPF and other progressive fibrosing ILDs include multidisciplinary management of patients from diagnosis to treatment and follow up, early referral to specialty centers, close monitoring for disease progression and the continuous update of information and management strategies as the field is rapidly evolving. Evidence from a prospective cohort study in IPF patients suggests that delayed referral to tertiary care centers is associated with increased mortality regardless of IPF severity . AccordiThere is an urgent need for guidelines helping clinicians in the management of ILD patients with the new treatment options. As pointed out in a series of suggestions on how to improve future guidelines for the diagnosis and management of IPF, recommendations for progressive fibrosing ILD should not be added to IPF guidelines as this may create additional confusion . Thus, gThe INBUILD trial has shown that when the progressive fibrosing phenotype develops, a definite diagnosis of the underlying ILD is not required to initiate antifibrotic therapy with nintedanib and that a working diagnosis is sufficient . This obThe use of combination therapy that combines, for example, antifibrotic and immunomodulatory agents may further improve the treatment of progressive fibrosing ILDs. Subgroup-analyses of the INBUILD and SENSCIS trials by immunomodulator use at baseline have suggested that nintedanib can be used in combination with glucocorticoid  and mycoIn the justification of a progressive fibrotic phenotype, the clinical and biological rationale must complement therapeutic decisions, with improved understanding on how to best define disease progression and, ideally, how to predict its occurrence prior to actual progression of the utmost importance . HoweverOur current understanding of the pathogenesis of progressive fibrosing ILDs is limited, and treatment guidelines, other than for IPF, are lacking. However, the results of trials investigating antifibrotic drugs in patients with fibrosing ILDs other than IPF, which have shown a reduction in the rate of disease progression, are considered a breakthrough in the field of ILD. Importantly, the INBUILD trial demonstrated that a definite diagnosis of the underlying ILD was not required to initiate antifibrotic therapy when the progressive fibrosing phenotype develops and that a working diagnosis was sufficient. Therapeutic decisions based on a working diagnosis are likely to increase the access of ILD patients to highly effective therapies and ultimately improve long-term outcomes. Ideally, an increasing understanding of the pathogenesis of progressive fibrosing ILDs, a clear definition of a progressive fibrosing phenotype and the identification of validated biomarkers to predict disease progression may direct treatment towards more precise and targeted options. Indeed, ongoing clinical trials with nintedanib and pirfenidone, as well as other novel antifibrotic agents under investigation, are expected to advance our understanding of the therapeutic potential of antifibrotics for the treatment of progressive fibrosing ILDs. Ideally, it is expected that the adoption of new drugs in clinical practice alongside treatment optimization will improve the management of patients with these rare conditions.


The demand for graphene-based devices is rapidly growing but there are significant challenges for developing scalable and repeatable processes for the manufacturing of graphene devices. Basic research on understanding and controlling growth mechanisms have recently enabled various mass production approaches over the past decade. However, the integration of graphene with Micro-Nano Electromechanical Systems (MEMS/NEMS) has been especially challenging due to performance sensitivities of these systems to the production process. Therefore, ability to produce graphene-based devices on a large scale with high repeatability is still a major barrier to the commercialization of graphene. In this review article, we discuss the merits of integrating graphene into Micro-Nano Electromechanical Systems, current approaches for the mass production of graphene integrated devices, and propose solutions to overcome current manufacturing limits for the scalable and repeatable production of integrated graphene-based devices. For over a decade graphene has been studied as a 2D material with numerous favorable properties including high strength and elastic modulus , high elThere are obstacles for graphene to be implemented in MEMS/NEMS devices because the mass production of graphene has repeatability and scalability issues . For exaIn this review, we will introduce several on-going research efforts focusing on increasing graphene production scale, speed, and quality. We will also highlight the current challenges for graphene to be successfully implemented in MEMS/NEMS device applications at a large scale and suggest how these difficulties can be overcome.2 bonded honeycomb lattice structure. The theoretical foundations of graphene (or ‘2D graphite’) were introduced by P.R. Wallace who sought to calculate the band structure and the physical characteristics of monocrystalline graphite [2 substrate. This process was repeated on the transferred films until only a single monolayer of graphene was exfoliated. In addition to the ability to isolate graphene in such an inexpensive manner, this work highlighted the impressive electronic properties of graphene. Andre Geim and Konstantin Novoselov were awarded the Nobel Prize in 2010 for the discovery of graphene and its groundbreaking properties.Graphene is an atomically thin 2D carbon material with an spgraphite . Howevergraphite . They weSince its discovery in a freestanding form in 2004, graphene has surpassed the expectations of researchers around the globe. Graphene has remarkable mechanical, electrical, magnetic, thermal, and optical properties. There are several review articles that discuss these properties in detail ,12,13,14−1. This corresponds to a Young’s Modulus of 1.0 ± 0.1 TPa assuming a monolayer thickness of 3.35 Å. Lee et al. also measured an ultimate tensile strength of pristine graphene of up to 130 GPa [The excellent mechanical properties of graphene can be attributed to the strength of the double carbon spng agent . Lee et  ribbons ,17.The initial excitement and popularity of graphene as an exceptional 2D material was a result of its unparalleled electronic properties . The higgraphene . Graphen V−1 s−1 , while seratures . The eleeratures .The ability to extract heat out of micro/nano-devices critical in many applications, including in MEMS/NEMS. The thermal conductivity of monolayer defect-free suspended graphene as measured by Balandin et al. was in the range of 4840–5340 W/mK . Seol etFor applications in integrated MEMS/NEMS and electronic devices it is important to understand the coefficient of thermal expansion (CTE) of graphene. The theoretical first principles estimates show that graphene has a negative CTE at temperatures up to 2500 K . Bao et The centrosymmetric geometry of graphene means that the piezoelectric effect is not present in pristine films . This inxNy membrane to be as high as 18,000, while those of conventional Si devices ranges from 30–40.The sensitivity of piezoresistive sensors increases with larger sensing area and lower membrane thickness, which can lead to orders of magnitude higher sensitivities for graphene sensors . GraphenMicroelectromechanical Systems (MEMS) have been extensively researched since the 1960s, and the commercial viability of MEMS devices has been enabled by the development of standard silicon surface micromachining techniques ,32,33,34A useful application of MEMS/NEMS devices are radio frequency (RF) switches used in mm-wave communication systems ,50,51,52Graphene field-effect transistors with high on-off ratios  are alsoHowever, realizing reliable elecromechanical coupling with high bandwidth and low-loss resonant structures is a major challenge in designing MEMS/NEMS for these applications ,64,65,66−5 e/Hz, respectively, for a resonant frequency of 35.8 MHz and Q factor of 100 at room temperature [Graphene resonators have been used as mass sensors ,70,71,72030 Hz/g ,84, whil018 Hz/g ,85. Addi018 Hz/g . For for018 Hz/g ,86. Buncperature .The piezoresistive property of graphene NEMS can be leveraged for pressure sensors ,88,89,90700 mbar . Biomedi700 mbar . Resonan700 mbar . The pre700 mbar  as shown700 mbar .Recently, graphene field-effect transistors have been implemented as pressure sensors. These pressure sensors are typically designed to cover a wide range of pressures for tactile sensing and it has been reported to measure pressure changes from 2.5 to 30,000 mbar .For sensing strain, Zhao et al.  fabricatApart from resonators and piezoresistive sensors, suspended graphene has also been used to propose nanoscale generators ,107, eneBefore graphene can be used in any commercial MEMS/NEMS devices described in the previous section, large-scale graphene manufacturing processes must be implemented. The majority of existing literature on characterizing graphene properties and MEMS/NEMS focuses on small, lab scale samples which can not be produced with enough throughput for industry. There are many promising approaches to growing and transferring graphene on a large-scale but first the major limitations of any such processes must be understood.Graphene was first isolated through mechanical exfoliation in 2004 . This pr quality . HoweverThe next major advancement in graphene synthesis was growth on catalytic metal foils. Li et al. pioneered this work with monolayer graphene on copper foils . Copper While CVD has potential for being scalable, there are still some downsides to the resultant films compared to exfoliated films, namely grain boundaries and wrinkles. Exfoliated graphene benefits from being inherently single crystal however CVD graphene has grain boundaries that appear when the independent domains begin to stitch together . Grain bGraphene holds promise as a mechanical material in Microelectromechanical systems (MEMS) due to its extremely high elastic modulus and strength. Elastic modulus of exfoliated and CVD graphene is similar in many cases, which is around 1 TPa or 340 N/m   and the 0−6 K−1)  or Ni (110−6 K−1 ) causes 10−6 K−1 , which bRipples, on the other hand, are smaller in scale and can be harder to measure. Surface adhesion forces can obscure ripples on substrates, therefore ripples are best observed while suspended. While they can occur for a number of reasons, all wrinkles and ripples effectively soften graphene’s elastic modulus since energy is being used to flatten out the out-of-plane features instead of stretching the membrane . Prelimisurfaces . Therefosurfaces .Finally, graphene grain boundaries aren’t necessarily shared with the metal catalyst grain boundaries. Single graphene grains have been shown to be continuous along steps, positive and negative edges, and positive and negative vertices . This isResearchers have found that standard process steps can significantly reduce the fracture strength and toughness of graphene. Specifically, etching copper in ferric chloride  then etching away the metal substrate. Once supportive polymer layer with graphene is cleaned and transferred onto Si substrate, the sample is dried and annealed to improve adhesion between graphene and the substrate. Afterwards, supportive polymer layer is removed using solvent. This process is first reported for graphene grown using CVD  and overIn general, polymer-supported wet etch transfer is widely used for numerous applications. It is a convenient way to process graphene and effectively smooths and flattens the graphene onto the substrate. However, removal of polymer after transfer can be challenging as any residual polymers left on the surface tends to deteriorate properties of graphene. According to Wang et al. , graphen V−1 s−1 .On the other hand, direct delamination, or dry-transfer, can be achieved by directly peeling graphene off of the growth substrate and relocating it to the target substrate. The transfer utilizes rate dependence of adhesion forces to overcome traction energy between graphene and the metal substrate . AdditioAs shown in Electrochemical delamination uses ion intercalation between polymer-graphene and growth substrate  to separate graphene from the substrate . This mePolymer-free transfer eliminates the process of coating polymer on graphene and directly etches away the metal substrate. The problem of obtaining graphene without any kind of supportive layer has been resolved by utilizing hexane which traps graphene between hexane and metal etchant during release process and between hexane and DI water during the transfer process . Other mThe main problem with having transfer included in the MEMS/NEMS graphene device fabrication is that yield is difficult to control. MEMS/NEMS graphene devices often have trenches or gaps between electrodes in order to enable the graphene to be suspend on the device but this requires alignment of the patterned graphene to the MEMS substrate. One of the methods to achieve this is pre-patterening the graphene structures so that they can be align-transferred to different substrates  as shownResearchers are looking into ways to approach the problem of successfully manufacturing high-quality graphene at a large scale. Currently, the biggest challenges for mass produced graphene integrated MEMS/NEMS devices is having high quality graphene in large production scale with high throughput. Therefore, the aspects we considered important for proposed solutions are the ability to grow graphene over large areas, shorter growth times, maintaining the highest graphene quality, and improving the yield of the fabrication process. In order to summarize how current graphene growth research is being performed, we have plotted data from numerous references of growth time of graphene compared to electron mobility and grain size of graphene. As shown in In this section, several active research areas will be introduced and discussed for further enabling of high-quality graphene for mass production. Mass production schemes discussed in this section includes graphene transfer process using roll-to-roll (R2R) processing, graphene growth and MEMS device fabrication on transition metal thin films for transfer-free process, controlled graphene growth on single-crystalline films, strain engineering on the substrate, and other approaches.One of the most convenient and effective ways to reduce the cost of graphene production is to implement conventional manufacturing methods. Roll-to-Roll (R2R) processing is a well-known, high throughput process of making electronics on a roll of metal foil or flexible polymer. A variety of processes are available for coating, patterning, deposition, and etching in R2R manufacturing which can be performed continuously on a roll for miles at a time . For larThe first method is to etch the catalytic metal foil and roll the graphene on polymer into a separate roll for further processing. This process is conventionally known as wet etch transfer where only metal etchant touches the metal foil while graphene and the polymer are physically unstrained. The strength of this process is that graphene is exposed only to chemicals with which graphene is chemically inert. The weakness of the process is that the metal foil is not reusable as it is being etched away during the transfer process. In addition, polymer backing must be still be removed from the graphene for many applications .The second method is known as dry peeling method where polymer-graphene layer is peeled off from the metal foil. Initially, graphene and the metal foil are bonded with Van der Wall’s force. Force of dry peeling must be higher than Van der Wall’s force between two surfaces in order to successfully delaminate the graphene from the metal foil. The force can be adjusted by the speed and the number of graphene layers peeled off from the metal foil ,177.The strength of R2R process is that the throughput can be high depending on the growth process. The rate at which graphene grows on Cu foil is determined by the precursor gas, the temperature of the CVD furnace, and surface reaction mechanism between the gas and the substrate for graphene growth. The speed of the graphene production through R2R production is superior to any other manufacturing technique currently in use for graphene transfer. Also, graphene characterization for quality inspection through R2R process is much easier via high-speed tip-based metrology inspection. This technique has been developed to inspect nanostructures over large surface areas; therefore, it has a potential to be utilized to observe graphene quality after the production . In addiHowever, in order to further improve cost-effectiveness, the yield of graphene devices, and the device performance, it is necessary to advance graphene production so that it is compatible within current semiconductor fabrication.The R2R transfer process provides superior speed of graphene growth over a large scale, but the process itself requires an additional step of transfer to the target surface. Adding another step to the process can be difficult for the application where the graphene has to be a suspend nanostructure. Graphene integrated MEMS/NEMS devices show higher mechanical and electrical performance when it is fully suspended compared to when it is operated on a substrate. However, graphene is exposed to capillary force during liquid-based transfer which often damages the graphene .Transfer-free fabrication is one of the fabrication processes introduced to eliminate a step of using polymer backbone , or PMMA) to transfer graphene and instead grow graphene on the desired substrate ,181. TraThere have been efforts to eliminate the transfer process by growing graphene on metal thin-films directly above the devices they’re destined for. Patterning thin film allows graphene to grow on specified area and this can eliminate the necessity of patterning graphene for the device ,89,179.R is the average grain size, t is the thickness of film, and Using transition metal thin-films as a substrate for graphene growth is indeed a promising approach for application in MEMS/NEMS devices as depositing thin-film can be integrated into conventional MEMS/NEMS fabrication at the wafer scale. Depositing and patterning transition metal thin-films can also selectively grow graphene . Howeverubstrate .(2)Rt>3Eliminating transfer step sounds solid and appealing for ideal graphene fabrication process, but directly growing graphene on thin transition metal film is not trivial. There are several obstacles required to overcome to successfully grow graphene directly on a Si/SiOntegrity ,201. Cu ntegrity . Howeverre of Cu . TherefoThermally stable materials such as platinum have been studied to improve the integrity of thin film during and after graphene growth. Pt is known to have a higher melting temperature and less lattice mismatch while maintaining self-limiting graphene growth due to platinum’s low carbon solubility. For this reason, thinner Pt thin film down to 100 nm can be implemented to successfully grow graphene which is difficult with Cu thin films .Studies on selecting appropriate adhesion layers have also been reported where common adhesion layers of Ni, Ti, Ta, and Cr are used to support 300 nm Pt thin film to grow graphene. When the catalytic and adhesion layers are exposed to heat during graphene growth, those layers diffuse into each other and form an alloy. Therefore, alloying properties between the adhesion layers and the Pt thin film is important as imperfect alloying prevents uniform graphene growth on the metal thin film layer. The best alloying adhesion layer has been found to be Ni which uniformly alloys with Pt thin film at high temperatures ,198. TheThe substrate also plays an important role handling heat and affecting quality of graphene grown on top. For example, Pt thin film deposited on TiOubstrate  as shownDewetting of a film can be prevented by either increasing film thickness or decreasing the wetting angle. As cost effectiveness of graphene growth process is inversely proportional to the film thickness, it is difficult to choose the appropriate film thickness for many applications. Therefore, we would like to introduce several ways to achieve low temperature growth in next section to provide alternative solutions to the obstacle.Low temperature growth of graphene is important to preserve the integrity of thin-film and devices that will be integrated with graphene such as MEMS/NEMS; however, it is not trivial to find appropriate ways to achieve low temperature growth. The first reason is that decomposition of certain carbon source gases require high temperature. Decomposition of common carbon source, methane or CHcatalyst . The seccatalyst . Also, gcatalyst .Several approaches to achieve low temperature graphene growth without sacrificing graphene quality have been attempted. Graphene growth utilizing plasma-enhanced/assisted CVD has shown to lower the growth temperature to 600 °C with low defect peak measured in Raman spectrum ,205. WitThe importance of the substrate for graphene growth has been known and emphasized as any non-uniform and defective substrate conditions tend to deteriorate graphene’s quality . As discThe effort to grow single crystalline graphene on foil has showed prominent progress for the last ten years focusing on increasing the grain sizes and reducing nucleation sites during growth. Grain boundaries in graphene are between two grains of graphene with different lattice orientations which produces incomplete carbon-carbon bonding . Carbon graphene ,211,212.graphene ,143, theThere are two approaches for growing single crystalline graphene over large areas. The first approach is single-seed growth. Single-seed approach starts graphene growth from single nucleation site and grows it until the whole transition metal substrate is covered. This approach has no graphene stitching from graphene from different grains. If multiple nucleation sites present, each grains will have its own crystal orientation, and these individually oriented grains have potential to form polycrystalline graphene when grain boundaries are formed. Downside of single-seed growth is that larger areas take longer to cover with graphene since the process time is governed by graphene growth rate from the nucleation site.Eliminating majority of nucleation sites is challenging as the sites can be determined by the substrate’s roughness and other contamination. Reducing the surface roughness and the contamination can be done by pre-treatment process on the surface. For example, typical ammonium persulfate (APS) or acetic acid has been used to remove oxidation and other impurities on Cu foil surface . ChemicaMulti-seed approach has been widely used in growing polycrystalline graphene. The strength of utilizing multi-seed growth is that graphene starts nucleation sites in multiple locations which results in covering the substrate at a much higher rate. The detailed growth mechanism is explained in As alternative solutions of using monolayer graphene grown from CVD for NEMS/MEMS devices, graphene oxide (GO) has been introduced to the field. GO can be produced in solution so it does not require a CVD process. Also, scalability of GO is feasible through modified Hummer method ,216. PreThe atomic structure of GO can be seen in rGO drum resonators has been successfully utilized as nanomechanical devices in wafer scale as shown in We have reviewed several approaches to overcome the current processing problems in manufacturing graphene MEMS/NEMS devices. MEMS/NEMS applications generally require suspended graphene structures, but current graphene growth and transfer has many limitations. Majority of graphene growth on foil must be transferred to another substrate. However, graphene may be contaminated or damaged during this transfer process. The polymers used in the process also generally leave residue during and after transfer which deteriorates mechanical and electrical properties of graphene. In addition, transferring graphene to another substrate often damages and wrinkles the graphene. These are extrinsic defects that are added on top of intrinsic defects of graphene from the growth process that must be accounted for to create repeatable devices. In order to minimize extrinsic defects, transfer processes need to be minimized and selective graphene growth on transition thin-metal films may become necessary. However, as graphene growth requires very high temperatures to produce high quality graphene, maintaining the integrity of thin-metal film is very important. Studies related to improving the integrity of thin-film in high temperatures have been conducted and proposed for feasibility of graphene growth on transition metal thin films that are less than 100 nm thick. Additional work has also been done to reduce the growth temperature to make graphene growth more compatible with conventional MEMS processing. Finally, engineering the substrate and growth process to produce single crystalline graphene at higher rates is a very important step for improved graphene-based MEMS/NEMS devices because the grain structure of the graphene has a significant effect of its performance in MEMS/NEMS devices. Therefore, while graphene shows great promise in MEMS/NEMS applications due to its electrical and mechanical properties, significant work still needs to be done on the manufacturing processes for integrating graphene into MEMS/NEMS devices before graphene-based MEMS/NEMS devicecs can become commercially viable.