Rsh) is also observed across transferred graphene using this improved process.In this work, an improved rosin transfer process is initiated. An anisole coating is introduced based on the rosin transfer process to reduce the residue particles on the surface of transferred graphene. Rosin/graphene and anisole/rosin/graphene samples are handled without baking and with baking at different temperatures, i.e., 100 °C, 150 °C, and 200 °C. Atomic force microscopy (AFM) and Raman spectroscopy are employed to characterize the surface properties of transferred graphene. The removal of the protective rosin layer and anisole/rosin layers without baking is found to be more effective and beneficial compared to the conventional PMMA transfer process. Furthermore, better results in terms of reduced surface roughness and residue particles are accomplished by introducing anisole in the improved rosin transfer process. Uniform and low sheet resistance ( Based on aforementioned rare properties, graphene has become one of the most promising alternatives for Si. All of these features make graphene to step into the new generation of technologies beyond the limitations of conventional semiconductor materials [The isolated two-dimensional 2D) nature of graphene has attracted tremendous interest due to its exceptional properties. However, these excellent properties are attributed to the isolated single-layer graphene. Such unique properties include mechanical breaking strength of ~ 130 GPa D nature  comparedaterials –8.The properties described above are mostly related to intrinsic graphene. In reality, to achieve these complex properties, large area growth of graphene is required. For the growth of graphene, chemical vapor deposition (CVD) method is an efficient and inexpensive process for producing large area single-layer graphene . HoweverE.ad), good support strength, and good solubility in solvents and the last guarantees the easy removal of this protective layer after graphene transfer. Recently, rosin (C19H29COOH), a small natural organic molecule, was reported to provide a good protective layer with low Ead (1.04 eV) compared to popularly used PMMA (Ead > 1.45 eV), a good supporting strength, and, more importantly, an easy removal in solvents due to the intrinsic property as a small molecule [To meet these requirements, the first need is to study the reason for impairments on the surface of graphene. The impairments mainly result from the removal of the protective polymer layer in solvents. The purpose of this polymer protective layer is to protect the graphene from folding, tearing, and cracks. A good protective layer should have low adsorption energy  by low pressure chemical vapor deposition (LPCVD) in a quartz tube furnace [4:H2 = 0.5:300 sccm) was flowed in the furnace at the same temperature/pressure for 50 min to grow thin crystalline film of graphene. After the synthesis, graphene samples were cooled down to room temperature (the flow of methane was stopped at 600 °C). However, the carbon dissolves in metal up to a few atomic percent; the use of non-carbide forming metals, e.g., Cu, Ni, and Pt, is preferred [The graphene samples employed here were grown on a 25-μm-thick copper (Cu) foil  dissolved in ethyl lactate was used because of high viscosity and good film forming ability. Note that the employment of rosin with concentration less than 50 wt. % usually leads to less viscous, smother, and low film forming ability which cannot offer sufficient support for graphene. The rosin/graphene/Cu samples were then placed in cleansing solution (HCl:H2O2:H2O = 1:1:1) for 50 s to remove the dust and the residues attached on the back side of Cu during the spin coating. The accessible graphene-copper face was then etched by immersing in a marble solution HCl (50 ml):H2O (50 ml):CuSO4·5H2O (10 g) for 1.5 h, leaving behind a pliant membrane of rosin/graphene suspended in the solution. The suspended membrane was transferred to DI water for 5 times to rinse residual etching solution. The floating flexible and fragile membrane was transferred on the SiO2 substrate with care and precision. A modified rosin transfer process was proposed to further reduce the polymer residues and to improve the quality of transferred graphene, where rosin/graphene/SiO2 samples were spin-coated with anisole at 500 rpm for 10 s and at 1200 rpm for 30 s. All samples were categorized into not baked  and baked at 100 °C, 150 °C, and 200 °C for 30 min. The supporting rosin layer is removed by acetone bath, while anisole is used in the improved rosin-enabled transfer process which was also then removed by acetone bath. All transferred graphene was characterized using the Raman spectroscopy at 532 nm excitation wavelength in air using × 100 objective to determine the quality of pristine and as-transferred graphene layer using the improved rosin-enabled transfer process. The AFM characterization is done in tapping mode using the Bruker Dimension Icon model at standard temperature and atmosphere conditions. The four-point measurement (Kelvin technique) is performed to measure the sheet resistance at random points on the 2 × 2 cm2 area of samples.Figure Rq) values of as-transferred graphene are also collected by scanning surface area of 10 μm × 10 μm. Compared to Rq values of 0.889 nm, 0.97 nm, and 0.992 nm for graphene baked at 100, 150, and 200 °C, the lowest Rq value of 0.668 nm occurs for the graphene without baking. This, however, points out that baking is not beneficial in achieving a low Rq value which is also desired for practical device application of graphene. This Rq value can be especially used as the quantification of surface morphology of transferred graphene. The water molecules trapped between pliant graphene membrane and SiO2 during pickup from DI water would rupture the graphene, hence producing cracks within the graphene. As a result, the Rq value increases with increasing baking temperature. It is, therefore, not recommended to bake graphene at high temperatures even if baking is good at reducing residue particles.Figure Rq values for graphene without baking and with baking at 100, 150, and 200 °C are 0.523 nm, 0.887 nm, 0.95 nm, and 0.98 nm, respectively. A relaxation to as-transferred graphene with the introduction of anisole may help in achieving the lower Rq value of 0.523 nm in the improved rosin transfer process than that of 0.668 nm in the rosin transfer process, while the lowest value for Rq in case of conventional transfer method using PMMA is 1.03 nm. In this improved rosin transfer process, it is again proved that the baking is not beneficial in achieving a low Rq value because of similar reason, i.e., cracks produced during the baking at high temperature. Note that compared to the Rq value of 1.03 nm in the PMMA transfer process, both the rosin and improved rosin transfer process show much smaller Rq values, which manifests the superiority of adopted graphene transfer processes in this work. Compared with Rq roughness, the maximum height of large residual particles (Rmax) is also an important parameter in the application of large area thin film devices, because it determines whether short circuit may occur in devices. Figure Rmax at room temperature, 100 °C, 150 °C, and 200 °C. The minimum value for the Rmax, i.e., 15 nm, is achieved at RT for anisole/rosin/graphene. This also confirms the advantage of improved rosin transfer process at RT.Figure Rq values and Rmax, the quality of as-transferred graphene deserves to be evaluated. In Fig. −1 (G), a primary in-plane vibrational mode, and 2676 cm−1, a second-order overtone of a different in-plane vibration (2D), are found. These peaks are adduced from a 532-nm excitation laser. The position and shape of these two peaks are prominent, clearly defining the material to be graphene. Also, the ratios of 2D band to G band intensities (I2D/IG) are 1.61 to 1.65, indicating the single layer of as-transferred graphene. The absence of D peaks in the Raman spectra for as-transferred graphene with baking at different temperatures confirms that the disorder is unlikely to appear using both the rosin and improved rosin transfer process. Also, no rosin- and anisole-related peaks are detected for all transferred graphene. The assumption no rosin- or anisole-related peaks was made on the fact that the Raman spectra appeared to be same after transfer process as those observed compared to the Raman spectra of pristine graphene grown on Cu. The appearance of D peak after transfer process in the baked sample shows the induced defects during the removal of rosin. Furthermore, the rosin residues after transfer process are very low. Therefore, rosin-related peaks are unlikely to appear in the Raman spectra of as-transferred graphene.Despite that the improved rosin transfer process is obviously advantageous in terms of residue particles and −1 demonstrates the doping of rosin-enabled transfer process; the described phenomenon has been reported previously in the literature [−1 which is the smallest compared to those with baking at high temperatures. These results mean that room temperature is favorable for achieving high-quality graphene during the rosin transfer process.Shifts in both the G and 2D Raman peaks of graphene are usually produced by a combination of strain and doping due to the interaction with the substrate or support layer during transfer process. It is known that the blue shift of both the G band and 2D band positions indicated p-doping of graphene. The entailed 2D peak upshift of ~ 6 cmterature , 25. The−1 which is a little bit lower than that in Fig. In Fig. Rsh) data are collected and illustrated in Fig. Rsh is measured at 5 points on each sample. The size of the sample is about 2 × 2 cm2 in order to get reliable results. In Fig. Rsh data for as-transferred graphene using the rosin transfer process at random spots are presented. As seen, for all graphene, scattered Rsh values in the range of 500–700 Ω/□ are found across the surface of as-transferred graphene. The lowest value of Rsh occurs for the graphene without baking which is also in good agreement with the observations from the Raman spectra. In Fig. Rsh values for as-transferred graphene using the improved rosin transfer process are shown. As seen, compared to Fig. Rsh is much better and the range of Rsh values is significantly narrower, i.e., 500–600 Ω/□. More importantly, the Rsh values in the improved rosin transfer process are generally lower than those in the rosin transfer process and the lowest Rsh value of ~ 500 Ω/□ also happens for the graphene without baking. Figure Figure −1, which is obviously lower than 38.18 cm−1 for transfer graphene using the rosin transfer process. In addition, as-transferred graphene using the improved rosin transfer process shows generally lower Rsh values of 500–600 Ω/□ than those of 500–700 Ω/□ using the rosin transfer process. The baking at high temperatures is found to exert marginal effects on residue particles and quality for as-transferred graphene which is thus not recommended. Achieved results in this work are ought to be helpful in advancing clean graphene transfer process in order to realize graphene-based devices of high performance in the future.In this work, an improved rosin transfer process is proposed for the purpose to reduce residue particles further on the basis of the rosin transfer process. The established improved transfer process is compared with the conventional PMMA transfer process. It is found that this improved rosin transfer process by the introduction of anisole is indeed advantageous in terms of significantly reduced residue particles as well as good quality of transferred graphene. This remarkable reduction of residue particles would rather be attributed to the capability of anisole as a strong solvent in collaboration with acetone. Anisole/rosin dissolves more easily than bare rosin in acetone which leads to cleaner graphene in this improved rosin transfer process. The FWHM value of 2D band for as-transferred graphene using the improved rosin transfer process is 35.79 cm


In this study, the effect of the argon, nitrogen, and hydrogen gases on the final properties of the reduced graphene oxide- hydroxyapatite nanocomposites synthesized by gas injected hydrothermal method was investigated. Four samples were synthesized, which in the first sample the pressure was controlled by volume change at a constant concentration. In subsequent samples, the pressure inside the autoclave was adjusted by the injecting gases. The initial pressure of the injected gases was 10 bar and the final pressure considered was 25 bar. The synthesized powders were consolidated at 950 °C and 2 MPa by spark plasma sintering method. The final samples were subjected to Vickers indentation analysis. The findings of this study indicate that the injection of argon, hydrogen, and nitrogen gases improved the mechanical properties of the nanocomposites. Injection of gases increased the crystallinity and particle size of hydroxyapatite, and this increase was greater for nitrogen gas than for others. Injection of these gases increased the rate of graphene oxide reduction and in this case the effect of nitrogen gas was greater than the others.  But still, it has poor mechanical properties such as intrinsic brittleness, low fracture toughness (fracture toughness of 0.28–1.08 MPa.m0.5), poor tensile strength, and weak wear resistance which have limited the scope of its applications [Calcium phosphates have been widely used in the medical field. Members of this family include hydroxyapatite (HA), Tetracalcium phosphate (TeCP), α- Tricalcium phosphate (α-TCP), β- Tricalcium phosphate (β-TCP), dicalcium phosphate dehydrate (DCPD), dicalcium phosphate anhydrous (DCPA), and octacalcium phosphate (OCP). Among these bioceramics, HA is less soluble in the biological environment and therefore suitable for orthopedic applications as an implant , 2. HA iications –31. Reseications –49.Different methods have been applied to synthesize hydroxyapatite and graphene nanocomposites so far. Among these methods, the hydrothermal process has received much attention because of its lack of calcination stage, excellent control and the ability to synthesize in situ. In this method, precursors containing calcium and phosphate ions are used as suppliers of the elements required for hydroxyapatite and graphene oxide as the graphene precursor. The high-pressure hydrothermal process results in the synthesis of hydroxyapatite and the reduction of graphene oxide . During the process, calcium ions first bind to the graphene oxide surface agents by ionic bond, and finally, in reaction with phosphate ions, hydroxyapatite is synthesized in situ on the surface of the graphene sheets. The synthesized powders are consolidated in various ways such as spark plasma sintering (SPS) and hot pressing (HP). Recently, gas injection into hydrothermal autoclave has been used to increase the rate of graphene oxide reduction. Increasing hydrothermal pressure by injecting gas also improves the mechanical properties of hydroxyapatite. Increasing the hydrothermal pressure in this method increases the crystallinity and crystallite size of HA. But the type of gas injected will affect the final mechanical properties obtained. Previously, researchers have shown that nitrogen gas reduces graphene oxide more than hydrogen gas –63.To investigate the effect of the type of injected gas on the final mechanical properties, three types of gas including argon, nitrogen, and hydrogen are considered in this study and the pressure of the gases was assumed to be constant. The synthesized powders were then consolidated by SPS and examined for mechanical properties. Characterization methods for synthesized powders and sintered samples include X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), high angle annular dark field (HAADF), energy-dispersive X-ray spectroscopy (EDS) and Vickers indentation technique. The results obtained in this study are expected to be very useful for similar researches.The chemicals used in this study include calcium nitrate tetrahydrate , diammonium hydrogenphosphate , ammonium solution , and GO . Figure For sintering of the powders, SPS method was used (Dr Sinter SPS-1050-CE). Details of the sintering operation have already been described in detail , 65.Table Eq. (112/300) and I300 are the intensity of the hollow between diffraction peaks of HA in the planes (300) and (112) and the intensity of the peak of HA in the plane (300), respectively. Eq. where υ11/300 and In this equation, d, θ, and λ are grain size, Bragg diffraction angle, and wave length of used X-ray (Cu), respectively. β and ε are full width at half height (FWHM), and crystalline lattice strain, respectively. Diamond 3.2, ImageJ, and Origin pro 2016 were used in this study for drawing shapes and graphs.Figure Scheme Figure Figure Figure Figure −1 is related to the HA structure (Fig. Figure ure Fig. b. Accordure Fig. c, d. TheFigure Figure The findings of this study indicate that injection of argon, hydrogen and nitrogen gases improved the mechanical properties of rGO-HA nanocomposites. Injection of gases increased crystallinity and particle size of HA, and this increase was greater for nitrogen gas than for others. Injection of these gases increased the rate of GO reduction and in this case the effect of nitrogen gas was greater than the others. The powders synthesized in this study included graphene sheets decorated with HA nanorods. The addition of graphene sheets made the mechanical properties of HA more uniform. Powder characterizations showed that injection of gases increased the HA-related peak intensities in XRD analysis, decreased the GO-peak intensities in FTIR analysis, and increased the ID/IG ratio in Raman spectroscopy. The results of this study could be useful for gas injection applications in hydrothermal autoclave.


Electrical performances of the fabricated graphene interconnects were evaluated, and the impact of temperature and size on the current density and reliability was investigated. The maximum current density of 1.18 ×108 A/cm2 was observed for 0.3 μm graphene interconnect on SiO2/Si substrate, which is about two orders and one order higher than that of conventionally used copper interconnects and CVD grown graphene respectively, thus demonstrating huge potential in outperforming copper wires for on-chip clocking. The drop in current at 473 K as compared to room temperature was found to be nearly 30%, indicating a positive temperature coefficient of resistivity (TCR). TCR for all cases were studied and it was found that with decrease in width, the sensitivity of temperature also reduces. The effect of resistivity on the breakdown current density was analysed on the experimental data using Matlab and found to follow the power-law equations. The breakdown current density was found to have a reciprocal relationship to graphene interconnect resistivity suggesting Joule heating as the likely mechanism of breakdown.Graphene interconnects have been projected to out-perform Copper interconnects in the next generation Magnetic Quantum-dot Cellular Automata (MQCA) based nano-electronic applications. In this paper a simple two-step lithography process for patterning CVD monolayer graphene on SiO Copper being the state-of-art interconnect material is facing severe challenges while being scaled down to nano dimensions due to its increased resistivity that is mainly because of its surface and grain boundary scatterings4 and also susceptibility to electromigration effect5. Moreover, copper as an on-chip clocking material requires high currents and large dimensions6 in order to generate the external field in MQCA. To illustrate, copper interconnects (CI) used in MQCA occupies more than 2000 nm in order to generate the required external field for data propagation between the nano-magnets7. Therefore CI impose limitations on the emerging nanoelectronic applications leading to extensive research in finding an alternate material that can replace the CI.Interconnects are going to play an important role in the next generation Magnetic Quantum-dot Cellular automata (MQCA) based nano-electronics14 can be envisaged as a potential interconnect which could replace copper. Moreover, graphene is projected to be an excellent candidate material for interconnects due to its high carrier mobility (2/V-s)15, ballistic transport16, high current carrying capacity17 and high thermal conductivity18. On the other hand, the length of the nanomagnets used in typical MQCA devices are more than 100 nm and therefore the underline interconnects should be in size ranging from more than 100 nm and less than the CI which are currently used.Thus in this context, graphene20 resulting in the maximum current density of 4 ×107A/cm2 and 1.2×107 A/cm2 on SiO2 substrate respectively. This is an order of magnitude less than the state-of-the-art Graphene Nanoribbon where the size is typically much less than 100 nm hence, it is not suitable as interconnects in MQCA22. Thus in an attempt to increase the current density in the CVD grown graphene, several hybrid structures have been reported27 with an additional overhead of significant increase in the fabrication complexity. To address these issues we introduce here a simple fabrication procedure of patterning graphene interconnects in the context of next-generation MQCA, still retaining an order of magnitude higher current density as compared to the present CVD graphene interconnects.CVD based multilayer and monolayer graphene interconnects have been investigated in2/Si substrate. Thus in this paper we report-a simple two-step lithography process for patterning CVD monolayer graphene interconnects resulting in one order of magnitude higher current density andeffect of temperature and size on the breakdown current density of CVD-monolayer graphene interconnects.Furthermore, in order to analyse the problem of integrating graphene interconnects in next-generation MQCA based nano-electronics, it is necessary to understand the effect of temperature and size impact on the electrical parameters. Graphene being projected as a potential alternative for interconnect material, the assessment of temperature and size would be of utmost importance. However, published literature lacks data on symbiotic effect of temperature and size on the electrical conductivity of graphene. Hence an organised attempt has been made here to understand the combined effect of temperature and size on the electrical behaviour of CVD grown monolayer graphene on SiOThe rest of the paper is organised as follows: Section 2 describes the experimental setup, section 3 describes the results followed by its discussions and the section 4 draws the conclusion.20. This results in the usage of different polymer resist that leaves behind the resist residues in each step of the lithographic process thus reducing the current carrying capacity of the graphene interconnects.State of the art, patterning of CVD based graphene interconnects involves a minimum of five lithographic processes i.e. two times optical and three times electron beam lithographyIn this paper, we report a novel two-step lithography process for CVD monolayer graphene patterning. A detailed description with pictorial representation has been given in Fig. 2 (300 nm thickness)/Si substrate using wet transfer process that was procured from M/s. Graphenea Inc. USA. The grain size was approximately 10 μm for monolayer graphene with greater than 95% coverage and with small multilayer islands.Initially, we started with graphene monolayer (10 mm × 10 mm) produced by CVD on copper catalyst and transferred to a SiO2. Figure 2 drying. Subsequently metal deposition was carried out at a pressure of 10−6 Torr and then lift off was done in acetone for 4 hours.The first lithography step involved patterning the metal contacts and the alignments marks. The alignment marks and the metal contacts Cr/Au (10 nm/70 nm) were fabricated by electron-beam lithography (EBL). Bilayer EBL resist, has been used in the reported experiment in order to achieve better liftoff of Gold (Au). PMMA - a high-resolution e-beam resist and Copolymer EL9 is selected as the bilayer resist material for the EBL. The sample is exposed to an electron beam of acceleration voltage 16 kV and a dose of 180 μC/cm2. The resist was developed in Microposit MIF319 developer and Tetra-methyl ammonium hydroxide (TMAH 2.3%) for 1 sec. While using HSQ an overlying dielectric layer is formed on the graphene interconnects. A low power oxygen plasma etch for 1 min 30 sec and at 50 Watt power was used, but still the fine line remained. After the plasma etch the HSQ resist pattern was etched into the graphene flake.The second lithography step involves patterning the graphene interconnects with width (W) in the range of 300 nm to 1500 nm, length of 1 μm and thickness of 0.335 nm. HSQ (Hydrogen Silsesquioxane) was used for patterning the graphene channels at 2000 rpm for 45 sec and exposing it to acceleration voltage of 18 kV and a dose of 190 s μC/CmThe Current-voltage (I-V) characteristics of the device were measured using Proxima (Fast IV Measurement/B1500) and Keithley 4200 Semiconductor Characterization System with two-probe configuration. The (I-V) characterisation was done by sweeping voltage in steps of 0–0.5 V, 0–1 V followed by 0 to breakdown voltage. Due to increasing current density in the graphene interconnects, there was a voltage at which the break down occurred in the graphene interconnect, resulting in a visible drop in current. The device testing was stopped at this point. This way of measurement helps in suppressing the effect of trapping centres due to resist residuals. Breakdown voltage for all cases was nearly found at 2 V. The temperature measurements were carried out at 298–473 K respectively. Temperature vs I-V characterisation results are discussed in the following section.22 that HSQ coating does not degrade the carrier mobility or rather they sometimes improve the mobility. Thus the reported two-step process of fabricating graphene interconnect not only reduces the rigour of fabrication but also reduces the residual trapping leading to higher current capacities of the graphene interconnects that is discussed in the subsequent section.However, it has been reported inSEM and Raman spectrum studies were conducted at several locations in order to confirm the uniformity of the monolayer graphene film.−1, respectively. This ensures I(G)/I(2D) = 0.55, which indicates that it is a monolayer graphene sample33. Figure 2 (300 nm/Si substrate).Figure 38. Therefore we have used CVD Monolayer graphene film on SiO2/Si substrate procured from M/s. Graphenea Inc, USA, with a current density of 102 A/cm2, having width of 1 Cm × 1 Cm × 0.35 nm as the starting material for patterning graphene interconnects.In spite of graphene devices that are produced from highly oriented pyrolytic graphite (HOPG), laser ablation, spin coating, CVD graphene is widely known method for device fabrication since it is by far the most popular way for producing graphene and also results in relatively high quality graphene, potentially on a large scale20 have also used CVD graphene for electrical characterization of the patterned samples and the maximum current densities reported are up to 4×107 A/cm2 and 1.2×107A/cm2 respectively which is about an order lesser than that of our fabricated graphene interconnects with the maximum current density of 1.18 ×108 A/cm2, which is attributed by the proposed simplistic two-step procedure.Besides, study reported in20.The major bottleneck for graphene application is, while patterning due to multistep process - the electronic transport properties gets degraded due to the resist residual trapped in the grain boundaries and on the surface of the CVD graphene that acts as scattering sites, limiting the transport of charge carriers resulting in the reduction of the electron mobility and also increase in the sheet resistance of the CVD graphene sheet20.Thus in this paper, a simple two-step lithography process has been reported for patterning graphene. As the number of lithography steps has been reduced, the effect of resist residuals and contaminants on the graphene surface also gets reduced resulting in one order higher current density as compared to other patterned CVD graphene current densities reported inThe results obtained has been divided into four main parts3 39, hence in order to place the easy axis of the nanomagnets on the graphene interconnect, the minimum width of the graphene interconnect was selected in the range of equal to or more than 300 nm and less than the copper interconnect, so as to maintain the keep-out zone .The variation of current with width 0.3 to 1.5 µm) of monolayer graphene interconnect is given in Table .5 µm of It was observed that with an increase in width of the graphene interconnects, the current also increased. It was found that the current (at breakdown voltage) increases at an increasing rate, though the breakdown current density decreases as shown in Fig. 40.The results obtained indicate that as the width increases the influence of the interconnect boundaries (rough area) decreases leading to lesser scattering and increased conductivity & current8 A/cm2, was observed which is an order more than state-of-the-art20, whereas the minimum was found to be 3.68 ×107 A/cm2 for 1.5 μm interconnect at room temperature.Breakdown current densities for all the cases were calculated and a maximum current density of 1.18×1021. The voltage and current were found to follow ohm’s law, i.e. V α I.This may be attributed by the reduction in the number of lithography steps as compared to the state of the patterning of CVD Graphene interconnects. Figure Patterning CVD monolayer graphene samples is quite an exhaustive, complicated and expensive process and thus we have formulated a mathematical expression for calculating the current from the width of the interconnects. The relationship between the current and width has been derived from Fig. The best fit for the obtained values of the intercept were found to be a = 143.93, b = −177.47 and c = 288.44. The graphene interconnects between the two metal electrodes on an insulating oxide layer was electrically characterised in the temperature range of T = 298 K to 473 K. The typical I-V characteristics of the CVD monolayer graphene interconnect of a 0.6 µm is given in Fig. ρ = ρ0 + AT (A is a positive constant). Therefore, it can be observed from the above figure, that the resistance dependence of temperature can be modelled by the equation.It was observed that with the increase in temperature, the current decreased. The resistivity was found to follow the law Temperature Coefficient of Resistance (TCR), defined as:The sensitivity to temperature was also calculated for this case by using the −1. Thus it was found that TCR was positive and therefore with increase in temperature, the current decreased.The temperature coefficient of resistance at 298 K for 0.6 µm was found to be 0.00248 KTCR for all other cases of the study was also calculated and is listed below:-i.e. the sensitivity of the resistivity to temperature variations decreases. The TCR for 1 µm was found to be about 38% larger compared to 300 nm wide interconnects.From Table The variation of current as a function of a) width of graphene interconnects, and b) temperature ranging from 298 K to 473 K are shown in Fig. We observed that interconnect of width 0.3 μm could not withstand the temperature of 473 K and rather failed after 423 K. The drop in current at 423 K for 0.3 μm width as compared to room temperature was noted to be 17% whereas, for all other cases, the drop in current at 473 K as compared to room temperature was more than 30%. The maximum drop in current was observed for 1.5 μm as 51%. Thus temperature was found to play a significant role in the current-carrying capacity of the graphene interconnect thus leading to decreased current densities.et al.41, that Cr/Au forms a good Ohmic contact with graphene and hence the contact resistance (Rc) due to the Cr/Au pads were found to be negligible as compared with the sample resistance. Similarly, as reported by Wei et al.42 the contact resistance versus contacts inter-spacing plot for CVD grown graphene with Cr/Au contact pads deposited on top of graphene channel was found to be 20 ohms for the contact inter-spacing of 1 µm. The similar observation on the contact resistance was also reported by Cai et al.43, where Cr/Au metal contact pads were fabricated on Single-layer graphene yielding 33.5 Ω which was found to be compared favourably with the study reported in42. We have adopted the procedures followed in the previously reported studies43, with a similar kind of setup for planning our experiments. The results shown in43 can also be correlated to the experimentally fabricated monolayer graphene interconnect used in our study.It has been reported in the literature by Withers 43, the contact resistance is within the range of 20–34 ohm. The total resistance of the proposed fabricated graphene interconnects falls in the range of 3.5–18 KΩ, and thus the impact of the contact resistance (in the order of 20–34 ohm) on the total resistance can be considered as negligible which is also aligned with the findings reported in17 by Murali et al.Moreover from Fig. 2 substrate on which graphene interconnect is directly lying.Figure 2, C and b are constants while 8 and b = −0.64 with ρ having the units of μΩ-cm. The effect of resistivity on the breakdown current density was analysed on the experimental data using Matlab. From the plot obtained Fig. , it was The exponent term of 0.64 represents the breakdown of the graphene interconnects with increasing resistivity, indicating that higher resistivity is responsible for the degradation of breakdown current density. This higher resistivity can be attributed to the defect morphologies such as the lattice imperfections, in-plane defects and voids, thus resulting in - jJoule’s heating that finally results in the electrical breakdown.x) along a conductor is governed by the heat equation –g is the thermal conductance of the GNR, and a is the area of cross-section. Inserting the values of k, g from ref. 17, the maximum temperature was found to be 1073 K (800 °C) at x = l/2, where l is the length of the graphene interconnect.The maximum temperature point of a suspended longitudinal dc biased graphene interconnect was found at the middle point. In steady-state conditions, the distribution of temperature θ, where l is the length of the graphene interconnect resulting thermal oxidation of graphite45 and maybe intimately connected with self-heating of the graphene interconnects.Thus the mechanism of breakdown reported here is believed to be Joule’s heating. Similar phenomenon has been reported by Collins Furthermore, in order to explain the constant breakdown voltage with the increment in width, the Power dissipation (Joule’s heating) for all cases was calculated as shown below:-It can be observed from Table Thus from the experimental observation, it can be found that:-It can be noted that, although the product of I and R, i.e., breakdown voltage (V) remains the same, for various widths, the quadratic component I play the dominant role in contributing to the self-heating phenomenon which is referred here as the Joule’s heating and the same can be represented using the inequality below and is also tabulated in Table 2R is causing the breakdown in the graphene interconnects which is the jJoule’s heating. Also it can be noted that with the wider graphene patterns, both the heat dissipation and heat absorption capability increases due to increase in the surface area. Since the radiative heat transfer is directly proportional to the surface area, increase in Joule’s heating due to the quadratic component of current (I2) is therefore compensated by the higher heat dissipation resulting in averaging out the effect of increase in the temperature of the patterned graphene sample and hence the breakdown voltage doesn’t get lowered down even for the wider graphene patterns.Thus, the quadratic component of the current I is not same throughout and therefore the Power dissipation, P = IHence from the experimental observations and the theoretical calculations, the authors intuitively conclude that the mechanism of breakdown is attributed primarily by the self-heating referred to as Joule’s heating.A simple two-step lithography for patterning CVD monolayer graphene has been used, thus resulting in lower complexity, cost-effective solution and higher current density as compared to the state-of-the-art fabrication methods for CVD graphene.8 A/cm2 (for 0.3 μm width) that is 100 times (106 A/cm2) more than the copper, resulting in outperforming copper-based interconnects.The fabricated graphene interconnects were found to have a current density of 1.18 ×10Based on our experimental observation, we formulated a generalised equation between the current and width of the CVD monolayer graphene-based interconnect that enables to calculate the current or width values without doing fabrication.The current at the breakdown point was found to decrease significantly with the increase in temperature, indicating a positive temperature coefficient of resistance. Further, as width of interconnects increases from 0.3 to 1.5 μm, the current at breakdown voltage was found to increase, at an increasing rate. This is due to decrease in scattering because of lesser contribution of boundaries, as the width of interconnects increases.It was observed that the graphene interconnects failed at the mid-segment of the interconnects due to incipient fusion at the breakdown voltage.The failure mechanism of the graphene interconnects was analysed. We found that the current density and the resistivity follows a power-law relationship signifying that higher resistivity of the interconnects were also responsible in the degradation of breakdown current density. This higher resistivity can be attributed to the defect morphologies such as the lattice imperfections, in-plane defects and voids, thus resulting in - joule’s heating that finally results in the electrical breakdown.2 substrate obeyed Ohm’s law.Monolayer graphene interconnects on SiOThe drop in current at 473 K for nearly all other cases as compared to room temperature was more than 30%.The temperature was found to have a profound effect on the graphene interconnects. Results show that while working with lower widths of the monolayer graphene interconnects at high temperatures pose challenges.The effect of width and temperature on the electrical properties of CVD monolayer graphene has been investigated experimentally. The outcomes of the study are summarised below:8 A/cm2 for 0.3 μm graphene interconnect on SiO2/Si substrate, i.e. two orders higher than that of conventionally used copper interconnects, Graphene has an enormous potential in outperforming copper wires.Thus with the maximum current density being 1.18 ×10


The majority of cases of dyspepsia are functional, with a very rare cause of organic dyspepsia being gastric sarcoidosis. Although gastric sarcoidosis is the most common gastrointestinal manifestation of sarcoidosis, it is asymptomatic in >99% of cases. This is a case of a 48-year-old with known pulmonary sarcoidosis who was diagnosed with gastric sarcoidosis after presenting with dyspepsia. Dyspepsia is one of the most common gastrointestinal problems encountered in the outpatient setting, affecting 7%-45% of the population worldwide . The majOur patient was a 48-year-old African American female with a history of sarcoidosis, depression, and vitamin D deficiency. Her sarcoidosis was initially diagnosed by bronchoscopy and endobronchial ultrasound-guided biopsy after the patient was found to have incidental pulmonary nodules about six years prior to this presentation. She had been treated with systemic steroids and methotrexate, and had been stable off medications for one year prior to presentation. During a routine healthcare visit, she reported new onset of nausea and heartburn for one month. She was started on pantoprazole 40 mg daily, which only slightly improved her symptoms. In view of her systemic disease and advanced age, she was referred for an esophagogastroduodenoscopy, which showed Los Angeles classification grade A esophagitis of the gastroesophageal junction Figure  and erosBiopsies of gastric antrum and body showed severe active chronic gastritis with non-necrotizing granulomatous inflammation Figure , with neThe patient resumed treatment with steroids and improved symptomatically, and she is now being followed outpatient.Dyspepsia is a constellation of upper gastrointestinal (GI) symptoms, including the presence of upper abdominal pain or discomfort with or without other symptoms such as nausea, belching, and vomiting. Less than 30% of cases of dyspepsia are found to have an organic cause. Most cases of dyspepsia are functional, as defined by the Rome IV criteria . These iSarcoidosis is a multisystemic disease characterized by the histological presence of non-caseating granulomas, which most commonly manifests in the lungs. Gastrointestinal sarcoidosis is rare and usually asymptomatic, with only 0.1%-0.9% of all cases displaying symptoms . The mosRegarding diagnostic approach to GI sarcoidosis, a high degree of suspicion is required as the diagnosis is made histologically with upper endoscopy and biopsy. The endoscopic appearance varies depending on the type of involvement, but can range from normal appearing mucosa to erosive, friable mucosa with nodularity and prominent folds, ulceration, or polyps. A deep mucosal or full thickness biopsy is recommended, as superficial biopsies may be non-specific. Tissue biopsy typically demonstrates the characteristic histologic findings of sarcoidosis: non-caseating granulomatous lesions with multinucleated giant cells. It is imperative to rule out infections and other etiologies of granulomas with appropriate staining. Management consists of immunosuppressive and symptomatic therapy in patients with symptoms; asymptomatic patients are usually not treated . The maiPatients with dyspepsia can have significantly decreased quality of life, although their life expectancy is normal. A large number of cases of dyspepsia remain uninvestigated, with their overall pooled prevalence estimated at 21% . Althoughttps://insights.ovid.com/crossref?an=00000434-201910001-02739) [This article has been presented as an abstract (1-02739) .A rare organic cause of dyspepsia is gastric sarcoidosis, which can have a variety of presentations and endoscopic appearances. It can be diagnosed by its characteristic biopsy appearance, and treated with systemic steroids when symptomatic. Our case highlights a rare and likely underdiagnosed cause of dyspepsia, the importance of maintaining a broad differential diagnosis, and appropriate use of upper endoscopy for evaluation of patients with new-onset dyspepsia.


In any ‘omics study, the scale of analysis can dramatically affect the outcome. For instance, when clustering single-cell transcriptomes, is the analysis tuned to discover broad or specific cell types? Likewise, protein communities revealed from protein networks can vary widely in sizes depending on the method. Here, we use the concept of persistent homology, drawn from mathematical topology, to identify robust structures in data at all scales simultaneously. Application to mouse single-cell transcriptomes significantly expands the catalog of identified cell types, while analysis of SARS-COV-2 protein interactions suggests hijacking of WNT. The method, HiDeF, is available via Python and Cytoscape. Significant patterns in data often become apparent only when looking at the right scale. For example, single-cell RNA sequencing data can be clustered coarsely to identify broad categories of cells , or analyzed more sharply to delineate highly specific subtypes  –3. LikewHierarchical community Decoding Framework (HiDeF).Guidelines for detecting robust patterns across scales come from the field of topological data analysis, which studies the geometric “shape” of data using tools from algebraic topology and pure mathematics . A fundapersistent  , a databWe also applied HiDeF to analyze a collection of 27 human protein interaction networks , 31. We In contrast to clustering frameworks, HiDeF recognizes when a community is contained by multiple parent communities, which in the context of protein-protein networks suggests that the community participates in diverse pleiotropic biological functions. For example, a community corresponding to the MAPK (ERK) pathway participated in multiple larger communities, including RAS and RSK pathways, sodium channels, and actin capping, consistent with the central roles of MAPK signaling in these distinct biological processes   and a set of similarity relations between these objects (edges). Examples of interest include networks of cells, where edges represent pairwise cell-cell similarity in transcriptional profiles characterized by single-cell RNA-seq, or networks of proteins, where edges represent pairwise protein-protein biophysical interactions. We seek to group these objects into communities (subsets of objects) that appear at different scales and identify approximate containment relationships among these communities, so as to obtain a hierarchical representation of the network structure. The workflow is implemented in three phases. Phase I identifies communities in G at each of a series of spatial resolutions γ. Phase II identifies which of these communities are persistent by way of a pan-resolution community graph GC, in which vertices represent communities, including those identified at each resolution, and each edge links pairs of similar communities arising at different resolutions. Persistent communities correspond to large components in GC. Phase III constructs a final hierarchical structure H that represents containment and partial containment relationships (directed edges) among the persistent communities (vertices).Consider an undirected network graph network modularity, as a function of community assignment of all objects [resolution parameter integrated into the modularity function can be used to tune the scale of the communities identified [G to community labels, ki is the degree of vertex i, m is the total number of edges in G, γ is the resolution parameter, δ indicates that vertices i and j are assigned to the same community by A is the adjacency matrix of G. To determine http://github.com/vtraag/louvain-igraph; version 0.6.1). Values of γ are sampled logarithmically between lower and upper bounds γmin and γmax at a minimum density such that for all γ there exist at least 10 nearby γ′ satisfying:Community detection methods generally seek to maximize a quantity known as the  objects . A resolentified , 42, 43,γ values satisfying the above formula are defined as γ-proximal. The sampling step was practically set to 0.1 to sufficiently capture the interesting structures in the data; it is conceptually similar to the Nyquist sampling frequency in signal processing [γmin = 0.001, which we found always resulted in the theoretical minimum number of communities, equal to the number of connected components in G. We used γmax = 20 for single-cell data  and s(b):To identify persistent communities, we define the pairwise similarity between any two communities γ-proximal resolution values, as communities within a resolution do not overlap. To represent these similarities, we define a pan-resolution community graph GC, in which vertices are communities identified at any resolution and edges connect pairs of similar communities having J > τ. Each component of GC defines a family of similar communities spanning resolutions, for which the persistence can be naturally defined by the number of distinct γ values covered by the component. For each component in GC larger than a persistence threshold χ, the biological objects participating in more than p% of communities represented by the vertices of that component define a persistent community.Pairwise community similarity is computed only for pairs of communities identified at two different H, a directed acyclic graph (DAG) in which each vertex represents a persistent community. A root vertex is added to represent the community of all objects. The containment relationship between two vertices, v and w, is quantified by the containment index (CI):w shared with v. An edge is added from v to w in H if CI is larger than a threshold σ (w is σ-contained by v). Since J < τ for all v, w (a property established by the procedure for connecting similar communities in phase II), setting σ ≥ 2τ/(1 + τ) guarantees H to be acyclic. In practice, we used a relaxed threshold σ = τ, which we found generally maintains the acyclic property but includes additional containment relations. In the (in our experience rare) event that cycles are generated in H, i.e., CI ≥ τ and CI ≥ τ, we add a new community to H, the union of v and w, and remove v and w from H.We initialize a hierarchical structure represented by H, which represents the hierarchy returned by HiDeF describing the organization of communities. The biological objects assigned to each community are expanded to include all objects assigned to its descendants. Throughout this study, we used the parameters τ = 0.75, χ = 5, p = 75. Note that since χ is a threshold of minimum persistence, the results under a larger value of χ′ can be produced by simply removing communities with persistence lower than χ′  are less robust to network perturbation . We found that combining HiDeF with node embedding resolved this issue and further improved the performance and robustness  to generate benchmark networks with two levels of embedded communities, a coarse-grained (macro) level and a fine-grained (micro) level. Within each level, a vertex was exclusively assigned to one community. Two parameters, μc and μf, were used to define the fractions of edges violating the simulated community structures at the two levels. All other edges were restricted to occur between vertices assigned to the same community , k = 10 (average degree), maxk = 40 (maximum degree), minc = 5 (minimum number of vertices for a micro-community), maxc = 20 (maximum number of vertices for a micro-community), minC = 50 (minimum number of vertices for a macro-community), maxC = 100 (maximum number of vertices for a macro-community), t1 = 2 (minus exponent for the degree sequence), and t2 = 1 (minus exponent for the community size distribution). The numbers of coarse-grained communities and fine-grained communities in each simulated network were approximately bounded by minC, maxC, minc, and maxc , and the sizes of communities within each level were set to be close to each other (as t2 = 1).Simulated network data were generated using the Lancichinetti-Fortunato-Radicchi (LFR) method   using the implementation at http://github.com/vtraag/louvain-igraph (version 0.6.1). For Infomap, we used the version 1.0.0-beta.47 from https://www.mapequation.org/, and set “Markov time” (the “resolution parameter” of Infomap) to 1 and other parameters to default. In general, these settings generated trees with two levels of communities. Note that Infomap sometimes determined that the input network was non-hierarchical, in which cases the coarse- and fine-grained communities were identical by definition.Some community detection algorithms include iterations of local optimization and vertex aggregation, a process that, like HiDeF, also defines a hierarchy of communities, albeit as a tree rather than a DAG. We demonstrated that without scanning multiple resolutions, this process alone was insufficient to detect the simulated communities at all scales , which contains two datasets generated with different experimental methods of separating bulk tissues into individual cells: FACS and microfluidic droplet. We applied HiDeF to the shared nearest neighbor graph parsed from the data files  provided in that study. All data normalization and pre-processing procedures have been described in the Tabula Muris paper [FindNeighbors function [FindNeighbors function in Seurat.Mouse single-cell RNA-seq data  [Identical analyses were applied to the FACS and the droplet datasets, respectively, yielding a hierarchy of 273 and 279 communities, respectively   retainingreedyModularityCut method in the Conos package was used to select N fusions in the original dendrogram, resulting in a reduced dendrogram with 2N + 1 communities . Here, we used N = 125, generating a hierarchy with 251 communities  that had been used in a previous community detection pipeline, NeXO [http://nexo.ucsd.edu/. A reference GO for human proteins was downloaded from http://geneontology.org/ via an API provided by the DDOT package [We obtained a total of 27 human protein interaction networks gathered previously by survey studies , 31, alone, NeXO . This co package .http://nexo.ucsd.edu/, with a robustness score assigned to each community. To benchmark communities created by hierarchical clustering, we first calculated three versions of pairwise protein distances , a one-way Mann-Whitney U test was used to test for significant differences between two sets of protein pairwise distances: (set 1) all pairs consisting of a protein in c and a protein in the sibling community of c; (set 2) all pairs consisting of a protein in each of the two children communities of c. The communities were sorted by the one-sided p value of significance that distances in set 1 are greater than those in set 2.Since all methods had slight differences in the resulting number of communities, communities from each method were sorted in decreasing order of score, enabling comparison of results across the same numbers of top-ranked communities. HiDeF communities were ranked by persistence. NeXO communities were ranked by the robustness value assigned to each community in the original paper . To rankC∗ and a set of computationally detected communities g(i) is the best match of Ci in xmeasures package  [We adopted a metric average F1-score  to evalueasures) .FindNeighbors function [6 edges, which is on the same magnitude as the original network (2.23 × 106 edges). We then applied HiDeF to this SNN graph with different combinations of parameters (Additional file HiDeF was directly applied to the original networks in most of our analyses of protein-protein interaction networks and compared with the results of hierarchical clustering following the network embedding techniques , 26. We function . The park 2.23 × 06 edges.Three hundred thirty-two human proteins identified to interact with SARS-COV-2 viral protein subunits were obtained from a recent study . This liAdditional file 1: Supplementary figures 1-9.Additional file 2. Review history.


DMS produced from the reduction of DMSO was not a significant process. Surface concentrations of total DMSO ranged from 2.3 to 102 nM with biological consumption of dissolved DMSO between 2.9 and 111 nM·d−1. Comparisons between 14C2-DMSO assimilation and dissimilation rates suggest that the majority of dissolved DMSO was respired (>94%). Radiotracer microbial consumption rates suggest that dissimilation of dissolved DMSO to CO2 can be a significant loss pathway in coastal waters, illustrating the significance of bacteria in controlling organic sulfur seawater concentrations.The marine trace gas dimethylsulfide (DMS) is the single most important biogenic source of atmospheric sulfur, accounting for up to 80% of global biogenic sulfur emissions. Approximately 300 million tons of DMS are produced annually, but the majority is degraded by microbes in seawater. The DMS precursor dimethylsulfoniopropionate (DMSP) and oxidation product dimethylsulphoxide (DMSO) are also important organic sulfur reservoirs. However, the marine sinks of dissolved DMSO remain unknown. We used a novel combination of stable and radiotracers to determine seasonal changes in multiple dissolved organic sulfur transformation rates to ascertain whether microbial uptake of dissolved DMSO was a significant loss pathway. Surface concentrations of DMS ranged from 0.5 to 17.0 nM with biological consumption rates between 2.4 and 40.8 nM·d It accorganisms ,11,12,13rganisms . Howeverp), which gets released into the dissolved phase through algal cell lysis caused by grazing, viral attack or autolysis, or exudation [d by bacteria, and some phytoplankton containing extracellular DMSP-lyases, typically maintains relatively low nano-molar concentrations [The dominant source of DMS is thought to be via the microbial (bacteria and/or phytoplankton) enzymatic cleavage of algal-derived DMSP ,16,17, axudation ,22. Rapitrations .d are attributed to photochemical and microbial oxidation of DMS [Roseobacter clade using an enzyme called trimethylamine monooxygenase [p) coupled with transformation to the dissolved phase via a number of pathways, including permeative diffusion, cell lysis, and as a byproduct of cell activity, have also been suggested [d remain essentially uncharacterised, although biological DMSOd consumption in seawater was previously observed [d loss pathways include bacterial consumption, reduction to DMS, oxidation to dimethylsulphone, and export via sinking particles [d [d to DMS during anaerobic respiration [Hyphomicrobium [Arthrobacter [Methylophaga [Dimethylsulfoxide is also an important ubiquitous reservoir of organic sulfur in the ocean, where the total pool of DMSO is often greater than that of DMS , and equn of DMS ,25. The xygenase ,27. Howeuggested . The funuggested . The marobserved ,31. Possarticles . Severalicles [d . DMSO reicles [d , and a vpiration ,35. Growicrobium , Arthrobrobacter ,37, and ylophaga . Howeverylophaga .d uptake and dissimilation to CO2 were significant DMSOd loss pathways. Our results suggest that dissimilation of dissolved DMSO to CO2 can be a significant loss pathway (for DMSOd) in coastal waters.Our objective was to employ a novel combination of stable and radiotracers in order to simultaneously determine seasonal changes in multiple dissolved organic matter sulfur transformation rates, and to ascertain whether microbial carbon DMSOSurface samples (≤10 m) were collected from the Western Channel Observatory long term monitoring station L4, situated ~13 km south west of Plymouth . Water samples were collected by the RV Plymouth Quest using 10 L Niskin bottles mounted on a rosette sampler, which also housed a Seabird 19 + CTD. Sub-samples were decanted into acid-washed brown glass bottles, sealed with ground glass stoppers with no headspace, and at in situ temperature for the ~2 h transit back to the laboratory. Temperature variability during transit was +1 °C, which is within the in situ diurnal variability at station L4.https://www.westernchannelobservatory.org.uk/). Chlorophyll a concentrations were determined through fluorometric analysis of acetone extracted pigments [Synechococcus sp. numbers were determined by flow cytometry (Accuri C6 instrument) on unstained samples based on their light scattering and autofluorescence properties [Seawater temperature was determined from the Seabird CTD, which has an accuracy of ±0.001 °C . The conpigments . Nutrienpigments ,42 and ppigments . Numbersoperties . Phytoploperties .−1 and cryogenically trapped in a PTFE sample loop submerged in liquid nitrogen before desorption using boiling water to GC). For dissolved DMSP (DMSPd), the purged DMS sample was transferred to a glass vial with 10 M NaOH and hydrolysed for 6–24 h (to convert DMSPd to DMS). Following hydrolysis, samples were analysed as above. For dissolved DMSO (DMSOd), ~10 mg cobalt-doped NaBH4 was added to the purged DMSPd sample (which reduces DMSOd to DMS) and purged for a further 10 min, with DMS analysis as previous.Concentrations of DMS, DMSP, and DMSO in seawater were analysed sequentially using a purge and cryotrapping system coupled with sulfur specific gas chromatography using a Varian 3800 gas chromatograph with a pulsed flame photometric detector (GC-PFPD) using methodology described by Simó et al. , Simó ant), which includes particulate DMSP (DMSPp) and a minor fraction from dissolved DMSP (DMSPd), 7 mL of whole seawater was pipetted into a glass vial with 1 mL 10 M NaOH, and left for 12–24 h for hydrolysis to convert DMSP to DMS. Then, 1 mL was carefully pipetted to a glass purge tower for extraction of DMS as above. For DMSPp, 7 mL of whole seawater was gravity filtered through a 25 mm GF/F filter. The filter was placed in a glass vial with 7 mL MQ and 1 mL 10 M NaOH, and left for 12–24 h for hydrolysis. Then, 1 mL was pipetted to the purge tower and analysed as previously. For particulate DMSO (DMSOp), ~10 mg cobalt-doped NaBH4 was added to the purged DMSPp sample to reduce DMSOp to DMS, and subsequently analysed by GC as above. The detection limit of the system was approximately 2.9 pmol S. The standard deviation of at least duplicate experimental samples was on average 6%, 10%, 9%, 9%, and 7% of the mean for DMS, DMSPt, DMSPp, DMSOp, and DMSOd, respectively. DMS standards for calibration were prepared from DMSP  in a 10 M NaOH solution in Milli-Q water. Typically, 4–5-point calibration curves were carried out twice per month during the sampling period, with an r2 for the resulting linear regression of ng sulfur versus square root of the peak area of typically ≥0.996.For total DMSP  by withdrawing ~30 mL and immediately gently filtering through a Millipore filtration unit containing a 25 mm GF/F filter directly into a 20 mL glass receiving syringe . This was immediately injected into a purge tower, and purged with high purity nitrogen at a flow rate of ~100 mL min−1 for 15 min directly into the proton transfer reaction mass spectrometer . This results in an exponentially decaying peak, allowing the total amount of DMS in a sample to be calculated by integration of the total peak area. Baseline levels were attained after 15 min of purging. Calibration curves were prepared using pure DMS . A primary DMS standard was prepared gravimetrically followed by dilution to produce a secondary standard, using gas tight vials. Five working (tertiary) standards were made up by dilution of the secondary standard in ultra-pure water in 100 mL glass syringes, to produce a 5-point calibration. For analysis, sub-samples of each standard were taken using 20 mL glass syringes without exposing the sample to the air, and purged and analyzed as above. Calibrations were performed on each sampling date. DMSPd and DMSOd were sequentially reduced to DMS after adding NaOH (DMSPd) and cobalt-doped NaBH4 (DMSOd) [n = 11, p < 0.001), suggesting that DMS concentrations derived from PTR-MS analysis were on average 4% lower than those from GC measurements.We report DMS, DMSP (DMSOd) ,47 into 3-DMS, 99 atom % d, Merck, Gillingham, Dorset, UK), DMSPd  and DMSOd  at ~10% of in situ concentrations into triplicate experimental bags. These and a control experimental bag (without any stable tracer additions) were incubated for 3–4 h in the dark at in situ temperature. During experiments, sub-samples were collected over 3–4 time points from each bag using a 50 mL glass syringe via the inlet on the Tedlar bag. Approximately 30 mL was withdrawn (ensuring no headspace or bubbles) at each time point and immediately gently filtered through a Millipore filtration unit containing a 25 mm GF/F filter directly into a 20 mL glass receiving syringe. This was immediately injected into a purge tower and analysed by PTR-MS as above. This technique allows the simultaneous quantification of DMS derived from DMSP cleavage and DMSO reduction, gross DMS loss , and net change in DMS concentrations . DMS production from DMS cleavage was determined as the rate of accumulation of d6-DMS from d6-DMSP, while DMS production from DMSO reduction was measured as the rate of accumulation of 13C2-DMS from 13C2-DMSO. Biological consumption of DMS was calculated from the rate of decrease in d3-DMS. Net change in DMS results from the rate of change of DMS, and thus gross DMS production, is calculated as net change in DMS plus biological consumption.Isotope tracer incubation experiments were also conducted using surface waters collected from station L4 between May and October 2014. On each date, approximately 350 mL seawater was siphoned directly into four acid washed and rinsed Tedlar bags without exposure to ambient air. We simultaneously added DMS , the biological consumption of DMSOd (from loss of 13C2-DMSO corrected for any conversion to 13C2-DMS), and the biological consumption of DMSPd (from loss of d6-DMSPd corrected for any conversion to d6-DMS and d6-DMSPd). For subsequent isotope DMSPd and DMSOd analysis at each time point, 20 mL was withdrawn from the Tedlar incubation bags using a glass syringe and placed immediately into a 20 mL serum vial containing two pellets of sodium hydroxide, which were immediately crimp sealed. These samples were stored in the dark at in situ temperature for between 4 and 8 weeks [d and DMSOd were sequentially reduced to DMS after adding NaOH (DMSPd) and cobalt-doped NaBH4 (DMSOd) into the purge tower and direct analysis by PTR-MS as previously. Concentrations of stable isotopes were determined via PTR-MS at m/z of 63, 65, 66, and 69 for unlabeled DMS, 13C2-DMS, d3-DMS, and d6-DMS, respectively (as this method of soft ionization within the PTR-MS adds a proton to each compound with no fragmentation of compounds). Final concentrations were calculated using standard curves. To scale the rate of tracer consumption or production to in situ values, the calculated rates were divided by the concentration of added tracer  and multiplied by the concentration of natural DMS, DMSPd, or DMSOd as appropriate [d, and DMSOd were calculated from the inverse of the rate constants for the loss of d3-DMS, d6-DMSPd, and 13C2-DMSOd, respectively.On two of the sampling dates (21 July and 26 August 2014), additional samples were also taken during the time course incubations for the determination of DMSO 8 weeks ,50,51. Bropriate . Biologi14C2-DMSO was added to each bottle and incubated in the dark at in situ temperature (no headspace). Tracer nano-molar  additions of 14C2-DMSOd (14CH3SO14CH3) were added to samples to determine microbial assimilation into biomass and dissimilation to 14CO2. Labelled 14C2-DMSO was purchased from American Radiolabeled Chemicals, Inc  with a specific activity of 30 mCi mmol−1 and a radiochemical purity of >99% (based on high performance liquid chromatography). A primary stock was made by diluting 52 µCi into 25 mL of 18 MΩ milli-Q water (2.1 µCi mL−1), and was stored in gas tight amber vials in the dark at 4 °C. Storage trials suggest <6% loss in activity over 12 months. Addition volumes of 14C2-DMSO to seawater samples were <1% of the sample volume incubation experiments.Seawater samples from the coastal station L4 were collected from Niskin bottles via acid washed Teflon tubing directly into the gas tight dark glass bottles . Labelled d carbon assimilation, a volume of 100 mL of the seawater sample was withdrawn from the bottom of the gas tight sampling bottles with a Teflon tube attached to a gas tight glass syringe. The tube was detached and the glass syringe attached to a Swinnex filter holder containing a 47 mm Supor 0.2 µm filter [n = 10, for ~34,000 DPM added per incubation). Samples and procedural blanks were filtered in a downward position with application of a very gentle pressure , so DMS release .d carbon microbial oxidation to 14CO2 (dissimilation) was determined in triplicate by pipetting 1 mL samples (each from replicate 305 mL gas tight incubation bottles) into 2 mL micro centrifuge tubes , and adding 0.5 mL of SrCl2.6H2O (1 M to precipitate the 14CO2 as Sr14CO3), 20 µL of NaOH , and 100 µL of Na2CO3  . The efficiency of the process assessed by mass balance of added 14C label was 96% ± 3% (n = 6). After centrifugation, the supernatant was aspirated and the pellet washed twice with ethanol (80%), resuspended in 1 mL of NaOH solution (~10 nM) that had been adjusted to a pH of 11.7, before addition of Optiphase HiSafe III scintillant to create a slurry. The samples were vortex mixed and stored in the dark for >24 h before being analysed on the scintillation counter. This period ensures that any chemiluminescence arising from interactions between the added NaOH and the Optiphase scintillant subsides [−1 .DMSOn et al.  for 14C subsides . Procedud were determined from linear time course experiments (refer to −1) was initially calculated from a ratio of the 14C counts collected on either the filter (assimilation) or as precipitated 14CO2  divided by the 14C2-DMSO spike (DPM mL−1). The apparent rate constant was multiplied by the in situ concentration of DMSOd to calculate DMSOd assimilation or dissimilation rates (nM d−1). All rate constants were corrected by subtracting killed sample counts. In addition, dissimilation rates were corrected to account for a minor  contribution from assimilation into particles entrained within the Sr14CO3 precipitate.Microbial assimilation and dissimilation rates of DMSOrefer to , where t−1 (3 cells mL−1), mainly comprising of Pseudo-nitzschia (0.8 × 103 cells mL−1) and large (≥4 µm) Thalassiosira phytoplankton cells  and Neoceratium lineatum , respectively. From flow cytometry analysis, the numbers of nanophytoplankton (2–20 µm), picophytoplankton (<2.0 µm), and Synechococcus ranged between 0.12 and 15.9, 1.46 and 41.5, and 0.15 and 62.0 × 103 cells mL−1, respectively, and showed peaks in abundance during September  and, ty−1 c, which eptember c. Total r months d.t concentrations averaged 13.5 ± 8.9 nM (June–October). However, there were noticeably higher concentrations during May . The total DMSO concentration in surface waters ranged between 2.3 and 102 nM, with on average ~56% in the dissolved phase , excluding the three maxima that occurred during June and September. Net DMS production (change in 12C-DMS with time) ranged between 0.0 and 10.5 nM d−1 (average 3.7 ± 3.4 nM d−1n = 12). Summing net DMS production and DMS BC yields gross DMS production rates of 2.7–42.9 nM d−1  ranged between 2.4 and 40.8 nM d−1 a. The av = 11 −1 b. DMS pr1 −1 −1, b, and wa6-DMSPd and 13C2-DMSOd on two dates in July and August (organic S transformations are summarized in d (loss of 12C-DMSPd) of 20.7 ± 8.8 and 29.2 ± 13.5 nM d−1 on 21 July and 26 August, respectively. We calculated biological consumption of DMSP as 75.7 ± 14.3 and 48.4 ± 15.6 nM d−1 for July and August, respectively  of at least 55.0 and 19.2 nM d−1 for July and August, respectively  of 8.1 ± 3.3 and 18.9 ± 11.3 nM d−1, and thus calculated that gross DMSOd production must be at least 15.1 ± 6.4 and 6.7 ± 16.2 nM d−1 for July and August, respectively  ranged between <0.01 and 0.49 nM d−1 during June–December July 2014, and was at a maximum during summer months (d to CO2 (dissimilation) was significantly higher and ranged between 2.7 and 111.0 nM d−1, with maximum rates during June . The ranges of DMS, DMSPt, and DMS/DMSPt presented here also agree with those found in a previously published seasonal cycle at L4 [Karenia mikimotoi , perhaps reflecting the ability of several species, for example, Scrippsiella trochoidea and Heterocapsa triquetra, to directly produce DMS by cleaving dissolved DMSP [Heterocapsa sp. on 23 June 2014 (118 cells mL−1), which may have also contributed to the DMS maxima observed at this time. The range in DMSPt concentrations found at the coastal L4 station is within the range often reported for a variety of other marine environments, including the North Sea, North Atlantic, Mediterranean, and subarctic Pacific [Phaeocystis sp. blooms (>200 nM) [p/DMSOp for our study was 5.4 ± 4.8, in agreement with the average reported in Simó & Vila-Costa [t concentrations remained lower than DMSPt concentrations, with maximum values not exceeding ~50 nM, as was similarly reported for coastal Antarctic waters [t values are within ranges reported globally [t , which corresponded to an over 140-fold increase in the number of dinoflagellate cells from a pre-bloom average of 0.91 to 131 cell mL−1. During this time, the dinoflagellate abundance was dominated (90%) by Heterocapsa cells, which were previously absent. We thus hypothesise that either there is direct production of DMSOd from the high-DMSP producing Heterocapsa [d  reduction under anaerobic conditions [d accounted for 55% of DMSOt and showed no obvious trends over the sampling period. The concentration of DMSOt in surface waters also showed a significant relationship with dinoflagellate abundance .The average surface DMS concentration found at L4 between May and October 2014 was 5.1 ± 4.0 nM, which compares well with the average found in U.K. shelf waters over the same monthly span of 5.4 ± 8.6 nM  and lowr et al. ). Surfacved DMSP ,58,59,60 Pacific , but did>200 nM) ,61,62. T>200 nM) ,30,56,62la-Costa . In compla-Costa . During globally ,30,32,47 to DMSO . This trseawater . The SARseawater , althougise DMSP . Growth nditions . Alternanditions  have rec−1), possibly because of lower temperatures minimizing bacterial activity [Scrippsiella trochoidea  and the diatoms Pseudo-nitzschia pungens  and Leptocylindrus danicus , where the numbers of phytoplankton cells varied between <0.04 and 7.68, <0.16 and 2.6, and <0.10 and 196.0 cells mL−1, respectively. The most noticeable was the peak in DMS BC during June, which coincided with an increase in the numbers of Scrippsiella trochoidea (from average background of 0.16 to 7.92 cells mL−1), which accounted for ~72% of all the dinoflagellates. Despite generally occurring in relatively low numbers . Generally, these rates are in the range previously reported for a variety of marine environments [d cleavage correlated with the abundance of the grazing ciliate Tontonia ovalis  and large flagellates , possibly suggesting enhanced DMSP lyase activity owing to grazing pressures, perhaps as some chemical “don’t eat me” cue [−1 of DMS could be excreted from biological particles  suggests a tight coupling in coastal waters.The biological consumption rates of DMS determined during this temperate coastal study were generally in the same range as those reported for coastal Antarctic  and subaactivity . Biologiactivity ,68,69. Or et al. ), Scrippllimolar  or 174–3t cell−1 . The bac to DMSO ,68, and ronments ,20,30,68 me” cue , or becad due to assimilation and dissimilation (determined using radiotracers) ranged between 2.9 and 111 nM d−1. However, the maximum loss rate was driven by the relatively high DMSOd concentration of 62 nM compared with the otherwise seasonal average of 9.2 ± 6.4 nM. Excluding the observed maxima, radiotracer-derived DMSOd biological consumption ranged between 2.9 and 24.3 nM d−1. Independent stable tracer experiments using 13C2-DMSO during July–August also suggest gross biological DMSO consumption rates of 23.3 ± 5.9 and 25.6 ± 11.6 nM d−1  at 0.6–1.9 d, which is comparable to 0.5–0.7 d derived from the 13C2-DMSO stable tracer experiments, and not dissimilar to previous literature estimates of 2–5 d [d turnover time of 7.4 d in coastal river plume stations, presumably because of their lower DMSOd oxidation rates. The majority of DMSOd utilized by the heterotrophic community was respired (>94%), like a variety of other low nano-molar organic methylated substrates in seawater such as methanol, methylamines, glycine betaine, and trimethylamine N-oxide [2 in the upper ocean [d respiration data lend support to this idea. Tyssebotyn et al. [Excluding the maxima, the biological DMSO loss rates (assimilation plus dissimilation) in coastal waters determined in our study averaged 11.9 ± 2.8 nM dubations ,76. By c9 nM d−1 , possiblof 2–5 d ,76. Howeof 2–5 d  report a N-oxide ,79,80,81 N-oxide . The micn et al.  similarld  and low DMS (1.0 nM) conditions  and 0.08 ± 0.04 (n = 3) µg C L−1·h−1 during July and August, respectively  to yield rates of 0.44 and 0.30 µmol C L−1 d−1 for July and August, respectively. This suggests that these three organic sulfur species could support 13%–17% of the estimated bacterial carbon demand during summer months. Bacterial carbon demand was converted to sulfur demand, assuming a carbon to sulfur ratio of 86 [−1·d−1 for July and August, respectively. Calculations suggest that DMSPd alone could supply all of the sulfur to meet microbial demand . Even if only ~50% of DMSPd sulfur used by bacteria was incorporated into biomass, that is, used for assimilatory rather than dissimilatory purposes, then the combined uptake of DMS, DMSPd, and DMSOd  would still meet bacterial sulfur demands. DMSPd is known to be a widespread substrate for heterotrophic bacteria, with literature suggesting that it can provide up to 15% and 100% of their carbon and sulfur requirements, respectively [d suggest that this compound alone could supply 60–126% and 1.4–2.9% of bacterial sulfur and carbon demand, respectively. Bacterial utilization of such reduced organic sulfur species over dissolved sulfate is thought to be energetically preferable for the synthesis of methionine [The rates of biological consumption of DMSPnditions . During pathways . Althougus cells c assimile et al. , where ue waters . DMS hasaters [d , with ou8 nM d−1 . We estit et al.  using a  al. [−1 ). Bacter−1 [0.58 . Bacteriio of 86 ,87, resuectively ,83. Simithionine .d, and its oxidation to DMSOd. Stable tracer experiments suggest that DMS produced from the reduction of DMSOd is not a common pathway in temperate coastal waters, which contrasts to Antarctic regions. Microbial consumption rates of organic sulfur species follow the order DMSPd > DMSOd > DMS, where the microbial dissimilation of DMSOd to CO2 can be a significant loss pathway for DMSOd in coastal waters. However, what controls the loss of DMSOd and the identification of bacteria responsible  in seawater largely remains elusive.In summary, these data demonstrate that, throughout the productive months of the year at a temperate coastal location, the biological consumption of DMS is highly variable, and largely decoupled from the amount of DMS produced from cleavage of DMSP


In addition, we tested a validation cohort including pulmonary sarcoidosis patients (n = 58) and healthy controls (n = 13). Initially, sera of three patients with sarcoidosis were screened using a protein array with 28.000 proteins against controls. Thereby we identified the Negative Elongation Factor E (NELF-E) as an autoantigen. With confirmatory Enzyme-linked Immunosorbent Assay (ELISA)testing, 29/82 patients (35%) with sarcoidosis had antibodies against NELF-E of the Immunoglobulin (Ig) G type, whereas 18/253 (7%) sera of the controls were positive for NELF-E. Clinically, there was an association of the frequency of NELF-E antibody detection with lung parenchymal involvement and corresponding x-ray types. NELF-E autoantibodies are associated with sarcoidosis and should be further investigated.Sarcoidosis is characterized by multiorgan involvement and granulomatous inflammation. Its origin is unknown and the potential role of autoimmunity has not been sufficiently determined. We investigated the presence of autoantibodies in sarcoidosis using protein array technology. The derivation cohort consisted of patients with sarcoidosis ( Sarcoidosis is a systemic disease of yet undetermined cause and is mainly characterized by multiorgan involvement with granulomatous inflammation ,2,3. BotDespite numerous attempts, only circumstantial evidence exists to regard sarcoidosis as an autoimmune disease, including its response to immunosuppressive therapy, association with certain Human Leucocyte Antigen (HLA) genotypes and detection of some autoantibodies that are also associated with other autoimmune diseases such as antimitochondrial, antinuclear, anti-double stranded deoxyribonucleic acid (DNA) and citrullinated cyclic peptide antibodies .Recent advances in protein array technology offers the opportunity for unbiased high-throughput screening of autoantibodies in sera. Protein arrays have been used repeatedly for the detection for autoantibodies in autoimmune diseases and malignant diseases . In the With the aid of this established screening platform using protein array technology, we identified autoantibodies in a cohort of patients with different types of autoimmune diseases or sarcoidosis. We found autoantibodies against the ribonucleic acid (RNA) binding protein Negative Elongation Factor E (NELF-E) significantly associated with sarcoidosis and pulmonary involvement. This is the first study demonstrating the presence of NELF-E antibodies in a specific disease. Further studies are needed to clarify the exact role of NELF-E autoantibodies in sarcoidosis.n = 24), patients with systemic lupus erythematosus , patients with febrile infectious diseases , patients who received a positron-emission tomography test , and healthy blood donors . Patients with sarcoidosis and SLE were recruited from the rheumatological clinic and hospital ward from 2011 to 2013. A second validation cohort of sarcoid patients was recruited from the interstitial lung disease outpatient clinic (n = 58) and healthy controls were recruited from our departments (n = 13). The study was conducted in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments were approved by our local ethical committee , and all patients provided informed consent.For the derivation cohort we collected the sera of patients with sarcoidosis  and World Association of Sarcoidosis and Other Granulomatous Disorders and SLE according to the former American College of Rheumatology criteria ,22. ChesClinical data were collected including available immunological data on antinuclear antibodies and extractable nuclear antigens, disease manifestation and course, histology, imaging, and treatment.Three different sera of patients with acute sarcoidosis presenting with Loefgren’s syndrome at disease onset were screened for the presence of autoantibodies by high-density protein array (human cDNA expression library (hEx1), Source BioScience LifeSciences, Nottingham, Nottinghamshire, United Kingdom) and subsequently compared against existing screens of patients with inflammatory or autoimmune disease. These patients were initially chosen due to their characteristic clinical features and brief disease history. The protein array is based on a cDNA of fetal brain tissue (Lib. No. 800). The clones were expressed by Escherichia coli. The screening was performed according to the protocol of Source BioScience LifeSciences. Sera of the patients were separately added to protein arrays. After overnight incubation, a second alkaline phosphatase labelled antibody goat anti-human Immunoglobuline G (IgG)  specific, Sigma Aldrich Corporation, St. Louis, MO, USA) in a dilution of 1:5000 was added to the protein array. Using a Typhoon 9400 scanner  and ECF  results could be imaged. The pictures were analyzed by Adobe Photoshop Creative Suite 3 . Subsequently, marked dots can be identified via an electronic library provided by imaGenes and compared to previous results in other diseases.For performing the ELISA tests, 96-well plates  were coated with 100 µg of the NELF-E peptide  overnight: Asn-Thr-Leu-Tyr-Val-Tyr-Gly-Glu-Asp-Met-Thr-Pro-Thr-Leu-Leu-Arg-Gly-Ala-Phe-Ser-Pro-Phe-Gly-Asn-Ile-Ile-Asp-Leu-Ser-Met. The next day, the coating solution was skipped and the plates were blocked with 300 µL per well 5% bovine albumin serum (BSA) for 1 h. The blocking solution was skipped and the plates were incubated with sera in a 1:100 dilution in 20% BSA phosphate-buffered saline (PBS) for 30 min at room temperature. For the definition of a cut-off, sera of 16 patients with sarcoidosis, 16 with infectious diseases and 16 blood donors were measured in duplicate. The optimal cut-off to differentiate sarcoidosis patients and controls was measured by Receiver-operating characteristics (ROC) analysis. The serum of a sarcoidosis patient which provided an optical density (OD) exactly at that cut-off was used as cut-off control in all further assays. The concentration of antibodies in this serum was defined as 1 (arbitrary unit) for IgG-type antibodies against NELF-E. After 30 min of incubation, the plates were washed 3 times with PBS. Next, 100 µL of a secondary peroxidase-goat anti-human IgG antibody labeled with horseradish peroxidase (HRP)  was added in a dilution of 2:10,000 or 2 µL in 8 mL PBS. The plates were incubated for 30 min at room temperature and washed 3 times with PBS. The color reaction was performed with tetramethylbenzidine (TMB)  for up to 15 min according to the manufacturer’s instructions and the ODs were read at 450 nm in an ELISA reader.p values refer to two-tailed tests. Two-tailed p < 0.05 was regarded as statistically significant. Analyses and graphs production were performed using STATA V16  RStudio version 1.2.5033 .Wilcoxon- and Kruskal-Wallis tests were used to evaluate differences in serum IgG concentrations and Fisher’s exact or Chi²-test were used to evaluate serum IgG cut-offs within disease groups and by clinical characteristics, as appropriate. All n = 24; second validation cohort n = 58), 43% of which were female with a median age of 48 years (interquartile range (IQR) 39–59). The majority of 88% had pulmonary involvement . Eighty-four percent had a chronic or relapsing disease with need for continued immunosuppressive therapy for at least 2 years. The remaining characteristics are displayed in We included a total of 82 patients with a definite diagnosis of sarcoidosis (first derivation cohort n = 4), CSS (n = 15), RA (n = 12), tuberculosis (n = 4), AOSD (n = 3), B-NHL (n = 4), Morbus Ormond/IgG4-related disease (n = 4), giant cell arteritis (n = 4).The sera of each patient with sarcoidosis bound to a number of autoantigens . One autIn the pooled cohort, 29/82 (35%) patients with sarcoidosis had antibodies against NELF-E of the IgG type  and 19/58 in the validation cohort (33%). 3/46 (6.5%) patients with febrile infectious disease (FID), 4/36 (11%) patients with systemic lupus erythematosus (SLE), 3/64 (4.7) positron emission tomography (PET) controls, 8/100 (8%) of blood donors (BD) and 0/13 healthy controls were positive for NELF-E .p = 0.041). The median antibody titer was significantly higher with lung parenchymal involvement (0.98 (IQR 0.89–1.02) vs. 0.77 (IQR 0.44–0.91), p = 0.0053) (Patients that had detectable anti-NELF-E IgG antibodies in the serum showed more involvement of the lung parenchyma compared to negative patients with regards to X-ray types (type 0 or 1 present in 4/17 (24%) anti-NELF-E positive patients vs. 25/64 (39%) (Fisher’s exact  0.0053) A and 4B. 0.0053) . We could show that antibodies binding to NELF-E of the IgG type are present in a subset of patients with sarcoidosis, particularly in those with lung parenchymal involvement, but not in controls including blood donors, systemic lupus erythematosus and other inflammatory/rheumatologic diseases. To our knowledge, the presence of NELF-E antibodies has not been described in the context of other diseases to date.While we recruited a fairly large number of sarcoidosis patients, only about 35% of them were found to produce anti-NELF-E antibodies. Recently, there were 5 clinical clusters described within the spectrum of sarcoidosis: abdominal; ocular–cardio–cutaneous–central nervous system; musculoskeletal–cutaneous; pulmonary–lymphonodal; extrapulmonary [NELF-E is part of the NELF complex. NELF-E shows the strongest RNA binding activity of the NELF complex and may start recruiting the NELF complex to RNA. NELF, a multisubunit complex containing RD, cooperates with DSIF to repress RNA polymerase II elongation ,25,26. NLittle is known considering NELF-E in autoimmune diseases or sarcoidosis. Hočevar et al. published recently a statistical analysis of genome-wide association study (GWAS). GWAS signals could be found increased in the region of NELF-E, SKIV2L, and STK19 genes . In our Sarcoidosis is not considered as an autoimmune disease but may have autoimmune features. In some patients, suffering from autoimmune diseases and sarcoidosis presence of autoantibodies have been also reported. Grunewald et al. identified lung-accumulated Vα2.3+Vβ22+ T-cell clones in association with HLA-DRB1*03 molecules, and inter-patient similarities between both T-cell receptor (TCR) α and β chain sequences which might indicate a specific antigen in sarcoidosis . BesidesIt remains unclear whether NELF-E autoantibodies have any functional consequences or are merely a bystander phenomenon. Preliminary, yet unpublished data, suggests that NELF-E autoantibodies may develop because of a mimicry effect between a certain part of the amino sequence of NELF-E and Mycobacteroides abscessus and other bacteria, which would fall in line with previously published associations between sarcoid and these pathogens . FurtherNELF-E autoantibodies are associated with sarcoidosis and should be further investigated.