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.


Graphene oxide/rubber composites were experimentally investigated for obtaining their thermal properties. Three kinds of the composite matrix material have been used: NBR, HNBR and FKM. The reduced graphene oxide in the form of crumped flakes has been applied as the filler influencing on thermal conductivity of the composites. Two values of graphene oxide weight concentration have been taken into account in the investigation. Thermal conductivity of the composites and basic matrix has been measured by the professional apparatus with the use of the guarded heat plate method. Before measurements the preliminary tests using the simplified comparative method have been performed. The results obtained, both from preliminary tests and using the guarded heat plate method, show an increase in thermal conductivity with increasing the reduced graphene oxide content in the composite. The experimental investigation allowed to determine not only the increase in thermal properties of graphene oxide/rubber composites compared to the basic matrix, but also the absolute values of thermal conductivities. Additionally, the SEM analysis showed that the tested composite samples contain agglomerates of the rGO nanoparticles. The occurrence of agglomerates could affect the composite thermal properties. This was noticed in the comparatively measurements of the temperature of different composites during the heating of samples tested. The maximum enhancement of thermal conductivity obtained was about 11% compared to the basis matrix of the composites tested. Thermal properties of the composites considered have been studied among others in7 where the authors reported the results of thermal conductivity investigations for the graphene oxide/rubber composites. The authors in2 present the results of measurements of thermal conductivity of bromobutyl rubber composites with the ionic liquid modified graphene oxide. Weight concentrations of the graphene oxide in the composites tested were: 1; 2; 3 and 4%. The authors reported a maximum one, threefold improvement in thermal conductivity with 4% content of graphene oxide, compared to the basic matrix. The dependence of the composites thermal conductivity on graphene oxide concentration was approximately linear. In turn the study3 includes the results of thermal conductivity measurements in the case of graphene oxide/rubber composite with the basic matrix of carboxylated acrylonitrile butadiene rubber. The authors investigated the graphene oxides with different oxidation degrees. The graphene oxide content in the composites considered was 4 phr (parts per hundred rubber). The authors reported the increase in thermal conductivity of the composite in the range from 10.1 to 27.2% depending on the graphene oxide kind. The article4 deals to the graphene oxide/natural rubber composite which was prepared by a novel in-situ method. The authors reported 36% increasing in thermal conductivity of the composite compared to the pure rubber. Despite the low content of the reduced graphene oxide (0.1 phr) the high enhancement of thermal properties has been observed. In turn the work7 deals to investigations of the interfacial thermal transport in graphene oxide/butadiene-styrene-vinyl pyridine rubber composites by molecular dynamics simulation. The authors concluded on the best oxidation degree of graphene oxide about 15%. For this value the highest thermal conductivity of the composites is achieved.The rubber is used extensively in many engineering applications. To improve the material properties rubber-based composites are formed. Both the natural rubber and the synthetic rubber copolymers are used as a basic matrix in composite materials. One of the rubber applications is the use as a material of the roller bearing seals. Nanoplatelets derived from different forms of carbon have recently attracted attention as an useful filler in rubber composites. The filling of the composite with nanoplatelets improves the mechanical properties of the roller bearing seals. Additionally thermal properties are also enhancement which contributes more intensive heat dissipation generated in the bearing. The different form of carbon are used as the filler in rubber-based composites. There are: graphene oxide13 included investigations in thermal conductivity of graphene/rubber composites or graphite and carbon black as fillers. In all cases considered the increase of thermal conductivity has been observed.The another above quoted articlesThe main goal of the present work was to determine the thermal conductivity of the selected reduced graphene oxide/rubber composites applied as a material on roller bearing seals. Because of practical applications of the materials considered the knowledge of their thermal properties is important and useful. Enhancement in thermal conductivity by adding the filler in the form of the reduce graphene oxide improves the heat rejection from the bearings. Matrix materials of tested composites were acrylonitrile butadiene rubber (NBR), hydrogenated acrylonitrile butadiene rubber (HNBR) and fluoroelastomer (FKM). Due to the heterogeneity of materials investigated, special attention was paid to the measurement technique. Thermal conductivity of the composites and the basic matrix has been tested with the use of the guarded heat plate method. Discussion on the measurements techniques for thermal conductivity of not homogeneous materials confirmed the validity of the method applied.3, the specific surface area of 266 m2/g, the specific electrical conductivity − 24 S/cm2. The scanning electron microscope (SEM) images of the graphene oxide nanoparticles are presented in Fig. The reduced graphene oxide (rGO) used as the filler influencing on thermal conductivity of the composites investigated has been purchased from Institute of Electronic Materials Technology, Warsaw, Poland. The considered graphene oxide has been reduced with sodium hypophosphite. The basic properties of the rGO included: the appearance as the grey to black powder, hydrophobic and stable in air, the bulk density equal to 0.019 g/cm2 carbon of C=C bonding11. Other peaks are much smaller what indicates the considerable deoxygenation by the reduction process of the graphene oxide1. The XPS spectra obtained is comparable with the another types of rGO also applied as thermal conductive filler in rubber graphene composites4.As can be seen the rGO material consists of thin, crumpled flakes with diameter of a few micrometers which forming the loose, disordered, crumbling structures. The X-ray photoelectron spectroscopy (XPS) of the rGO considered is shown in Fig. 15 depending on the form of GO. In the case of the rGO used in the investigations the value of thermal conductivity has been estimated on the basis of the model of the effective thermal conductivity equation for two-component material. The results obtained have been included in Chapter 3—Results and discussion.The reduced graphene oxide applied as the filler in the rubber composites has a high thermal conductivity. The literature data gives values of GO thermal conductivities from several to over two thousands W/(mK)mBM is a mass of basic matrix.As the basic matrix of composites three types of rubber have been used. There were: acrylonitrile butadiene rubber (NBR), hydrogenated acrylonitrile butadiene rubber (HNBR) and fluoroelastomer (FKM). For the fabrication of rGO/rubber composites the conventional mechanical method enabling the relatively uniform dispersion of the rGO flakes in the rubber matrix has been used. The suitable amount of rGO in the form of crumpled flakes were directly dispersed to the basis rubber during the rolling process with the use of two-roll mill calendaring. The process has been performed on the laboratory roll-mill of 400 mm width and 150 mm diameter cylinder at temperature 70 °C for about 10 min. The two-roll mill applied has provided the most homogenously disperse the reduced graphene oxide in the rubber examined. After the rolling process the weight of the composite formed has been determined and the weight concentration of rGO has been obtained from the equation3. Before thermal investigations the experimental research on some mechanical properties of the composites considered have been performed. As the results the values of indentation hardness and elastic modulus have been received. Mechanical properties were investigated with the use of Ultra Nano Indentation Tester. The basic characteristics of the composites tested are included in Table The density of the graphene oxide/rubber composites have been determined using the volumetric weighing method. An accuracy of density measurements was estimated at ± 0.003 g/cmA chemical characterisation of the composites have been performed using the energy-dispersive X-ray spectroscopy (EDS). The samples for EDS analysis have been prepared directly from the rGO/rubber composites. Before investigations the external surfaces of samples tested were cleaned and degreased in ultrasonic bath of ethanol. Figure As can be seen in Fig. The next test enabling the characterization of rGO/rubber composites was the scanning electron microscope analysis of the cross section of the materials tested. Before investigations the samples were cleaned and degreased in ultrasonic bath of ethanol. Figures Scanning electron microscope observations of the filler dispersion and fracture surfaces have indicated that in the case of NBR and HNBR composites the relatively uniform distribution of reduced graphene oxide flakes occurs. However, there are agglomerates and their clusters. In the case of HNBR matrix material, where the hydrogenation process increases temperature resistance, the rGO flakes are less visible than for NBR. This makes it difficult to analyze the distribution of nanoflakes.In turn, in rGO/FKM rubber composites, the rGO flakes are even less visible, especially at low magnifications. The compaction of the flakes at the same rGO concentration is much smaller than in the case of NBR composites. This may indicate the presence of a large number of agglomerates of significant size.Measurements of thermal conductivity of the rGO/rubber composites with different graphene oxide contents were carried out in two stages. In the first stage, as the preliminary investigation, the comparative method has been performed. The obtained results encouraged further research. The second stage concerned the main measurements using the professional procedure.The preliminary investigations of thermal properties of the rGO/rubber composites had a qualitative character. They were performed on the test stand which has been constructed especially for this purpose. A view of the stand applied is shown in Fig. d = 29 mm and thickness δ = 13 mm. Such dimensions gave a relatively large sample volume which ensured that it can be assumed the same thermal properties in all sample volume, even if the distribution of reduced graphene oxide flakes is not uniform. Temperature detectors have been placed at the depth of 5 mm. The first stage of the experiment was to investigation the changes in time of the sample temperature during the sample heating. Measurements have been performed for the rubber being the basic matrix and for the rGO/rubber composites with different graphene oxide content. The comparative analysis showed the filler influence on thermal conductivity of the composites.The tested samples had a circular shape with diameter The second stage of the experiment was to determining the thermal conductivity of samples tested with the use of standard method on the professional apparatus.16, transient plane source method8 or transient hot wire method17. The alternative method applied in composite thermal conductivity investigations is a molecular dynamics simulation7. The laser flash method enables thermal diffusivity obtaining, therefore, for determine the thermal conductivity the specific heat and density measurements are necessary. The small size of a test sample is an additionally challenge both in diffusivity and heat capacity measurements. In the case of laser flash method the sample dimensions are about δ ≈ 1 mm, d ≈ 12 mm. In turn, measurements of the specific heat with the use of differential scanning calorimeter require samples of about 10 mg weight. In this case, if the material tested is heterogeneous, test results could be unreliable. In turn, the transient methods, both the plane source and the hot wire, require the infinite in all direction and isotropic test sample20. Ensuring these conditions is very difficult. The authors in4 applied the plate-shaped samples of rGO/natural rubber composite with side of 254 mm and thickness of 50 μm. Such dimensions approximate infinity in only two directions. The very small thickness of the sample could significantly affect the measurement results. The second transient method—the hot wire—requires placing the probe being the linear thermal source in the test sample. Unlike the previous method, the heat source has a relatively large dimension and in this case, if the tested material is solid, the minimizing of thermal contact resistance between the probe and the material tested is an additional difficulty. Therefore, the hot wire method is rather used for thermal conductivity measurements in liquids or bulk solids26. The another method used in thermal conductivity research is the steady state technique—guarded hot plate method (GHP)—when material sample that is analyzed is in complete thermal equilibrium. The guarded hot plate method is widely used in measurements of thermal conductivity of insulation materials, that materials characterized by high thermal resistance. Examples of recent works are29. The rGO/rubber composites, being the subject of the present work, are also characterized by high thermal resistance. Generally, the rubber—matrix of composites tested—is a poor heat conductor. It can be expected that the addition of graphene oxide will not cause very significant changes in thermal conductivity. This has been confirmed in preliminary studies. Thus it seems that the guarded hot plate method is right in rGO/rubber composites research. An additional advantage is the method accuracy. It is in the range from 3 for 8%. In the present research the authors determined the expanded uncertainty of thermal conductivity at about 6%. Comparing with the another technique—the laser flash method—an accuracy of the GHP method will be better. In the case of laser flash method the uncertainty of thermal diffusivity, specific heat and density measurements increase the uncertainty of thermal conductivity measurement.The literature reviewing sees that nowadays, in the measurements of thermal conductivity of rubber composites, the researches use techniques: laser flash method3.The GHP has been used for measurements of graphene oxide/rubber composite thermal conductivity also by the other researchersδ = 7.65 mm for N0, N1, N2 and δ = 7.67 mm for H0, H1, F1, F2; d = 50.8 mm for all samples. It has ensured obtaining the averaged values of thermal conductivity in the entire volume of the sample. Additionally, the thermal contact resistance between test sample and the apparatus plates  could be easily minimized. This is possible thanks to apply a compressive load on the test stack consisting of material sample and the heating and cooling plates.In the rGO/rubber composites considered in the present study, the distribution of reduced graphene oxide flakes is not uniform. Agglomerates are present in the tested material, thus, for the obtain reliable results, a relatively large material sample should be tested. Dimensions of the circular samples investigated were: 30. The instrument applied is commonly named as a guarded hot plate apparatus (GHP). Figures Thermal conductivity of the rGO/rubber composites with different graphene oxide contents was measured by the guarded heat flow meter method with the use of the Unitherm 2022 thermal conductivity apparatus. Measurements have been performed according to the Standard31.The GHP technique is widely taken as a primary method to determine the effective thermal conductivities of materials with relatively high thermal resistance. The GHP apparatus applied was consisted of a flat-plate test sample, a hot-surface assembly and a cold-surface one. The cold and hot surface assemblies have been placed above and below the test sample, respectively. The thermal interaction of the apparatus with the surroundings was prevented by the insulation material. The test sample of the composite material was held under a reproducible compressive load between two metal surfaces, each controlled at a different temperature. The lower contact surface was a part of a calibrated heat flux transducer. During the heat flow from the upper surface through the composite material to the lower surface, an axial temperature gradient was established. The measurement of the temperature difference across the sample along with the output from the heat flux transducer and the known sample thickness enabled determining thermal conductivity of the composites investigatedThe thermal resistance of a composite material sample has been obtained using Fourier’s law in a steady state. Assuming thermal equilibrium the Fourier’s law applied becomesRs—thermal resistance of the composite material sample, Tu—upper plate surface temperature, Tm—lower plate surface temperature, q—heat flux through the sample, Rint—total interface resistance between sample and surface plates.δ is a thickness of the sample tested.In turn thermal resistance is defined asq has been determined by measuring the appropriate temperature difference across the reference calorimeter according toTL is the bottom heater temperature and N is the reference calorimeter heat transfer coefficient.The heat flux q obtaining and the value of resistance Rint in Eq. /2 was in the range 25–26 °C. Temperature measurements have been performed with the use of the system of thermocouples type K which was the element of the applied GHP apparatus. The uncertainty of the temperature measurement method was equal to 2% while the uncertainty of the GHP method for thermal conductivity evaluation was 3%. Expanded uncertainties of thermal conductivity and temperature measurements have been estimated according to33. They were about 6.5% and 4% for thermal conductivity and temperature respectively.As in the preliminary investigations the thermal conductivity measurements have been performed for the rGO/rubber composites included in Table As an example, the detailed results of four tests performed for N1 material sample are included in Table Measurement results for all composites tested with the use of GHP apparatus are summarized in Table The investigation results received show that the addition of the reduced graphene oxide into different rubber matrix causes an increase of thermal conductivity. The visible changes in thermal conductivity are rather small and they are in the range of measurement uncertainty. However, the preliminary investigations in which the thermal regular regime characterized by the constant samples temperature differences has been achieved, confirm the influence of rGO addition on thermal conductivity. The maximum difference is observed in the case of rGO/NBR composites: N0 and N2, and it is about 11%. This result is comparable to the preliminary tests basing on the thermal regular regime theory. In turn the HNBR results are the same for H0 and H1 samples. However, the expected increase in thermal conductivity depending on graphene oxide content may be within the measurement error. It is similar in the case of FKM, where the differences in thermal conductivity are in the order of 4%.34 (Maxwell–Eucken) is given by the effective thermal conductivity equation in the formOn the basis of the results received for rGO/rubber composites thermal conductivity of the reduced graphene oxide applied has been estimated. Two classical models of effective thermal conductivity of two-component materials have been used. The first—ME model34 (Effective Medium Theory) is given byThe second EMT modelkef represents thermal conductivity of the composite material, subscripts 1 and 2 represent continuous phase of the material and the dispersed phase, respectively.In both mentioned above models φ1 and φ2, thermal conductivity of the rGO – k2 has been calculated. Application of the model ME gave the result k2 = 34.3 W/(mK). In turn from the model EMT—k2 = 21.1 W/(mK). The results received are in a good accordance with the literature results35 obtained for the graphene oxide with the use of non-equilibrium molecular dynamics method.Basing on the thermal conductivity of the N0 and N1 samples, using the formulas  and 6)  6) and tThe use of classical structural models to determine thermal conductivity of rGO validated the thermal conductivity measurement method applied.In the present study, investigations of thermal properties of the reduced graphene oxide/rubber composites have been performed. As matrix materials NBR, HNBR and FKM rubbers have been used. The graphene oxide reduced with sodium hypophosphite has been applied as a filler material improving mechanical and thermal properties of the composite. The composites were investigated in terms of applications as a material of the roller bearing seals. Before thermal investigations the experimental research on some mechanical properties of the composites considered have been performed. The results of the investigations showed an improvement in elastic modulus and indentation hardness of the rGO/NBR and rGO/HNBR rubber composites with the increase of rGO concentration.Thermal conductivity of rGO/rubber composites increases with rGO concentration increasing, the maximum enhancement occurs in the case of NBR matrix material.Due to heterogeneity of the composites tested the measurement method used in investigations is very important. The size of the material sample is significant.The another important factor determining the correctness of results is taking into account the measurement uncertainty. In the present study the differences in some results received are in the range of the measurement uncertainty.Thermal conductivity results received with the use of guarded hot plate method are consistent with the results from comparatively method based on thermal regular regime. Thus, the results can be regarded as reliable.The improvement of the rGO/NBR thermal conductivity compared with the pure rubber is comparable with the literature results received by guarded hot plate method for the same graphene oxide content. The received compatibility concerns the results with taking into account measurement uncertainty.The use of classical structural models for two-component materials verifies the measurement method applied in the present work to determine thermal conductivity of rGO/rubber composites.Thermal conductivity of the composites has been measured with the use of guarded hot plate technique. Detailed calculations of the measurement uncertainty of the method applied have been carried out. The preliminary qualitative investigations by comparatively method have been also performed. SEM analysis has been made and it showed that the tested composite samples contain agglomerates of rGO nanoparticles. On the basis of the studies conducted and the obtained results some conclusions can be formulated:


Chemical reaction with diazonium molecules has revealed to be a powerful method for the surface chemical modification of graphite, carbon nanotubes and recently also of graphene. Graphene electronic structure modification using diazonium molecules is strongly influenced by graphene growth and by the supporting materials. Here, carrying on a detailed study of core levels and valence band photoemission measurements, we are able to reconstruct the interface chemistry of trimethoxybenzenediazonium-based molecules electrochemically grafted on graphene on copper. The band energy alignment at the molecule-graphene interface has been traced revealing the energy position of the HOMO band with respect to the Fermi level. In pristine graphene, in fact, the Fermi energy separates the occupied and empty states at the Dirac point thus making it a gapless semimetal with a low density of states at the Fermi level. Due to the small conductivity of graphene in this condition, various approaches are being explored to change the charge carrier concentration such as the direct doping of graphene through the chemical modification of graphene itself, either by the introduction of defects or by elemental substitution of the carbon or through molecular adsorption. At the same time, an electronic bandgap for graphene, mandatory to develop graphene-based electronic devices, can also emerge as a result of the interaction with the substrate or the introduction of atomic doping, as well as of the presence of organic molecules on the graphene layer7. Nowadays, methods of graphene chemical modification includes the covalent attachment (or grafting) of aryl groups onto the graphene surface, which transforms the sp2 carbon atoms into the sp3 hybridization state9. With respect to the adsorption  of the organic molecules on the graphene layer, the functionalization by covalent bonding is efficient in the graphene electronic structure modification1.Being simple and scalable, chemical modification is becoming a promising approach to modify the graphene electronic structure2 carbon atoms of graphene forming a covalent bond. The reduction reaction consists of an electron transfer to the aryl diazonium ions, releasing molecular nitrogen and creating a reactive intermediate aryl radical. This radical usually reacts directly with the carbon surface or can undergo polymerization with other aryl radicals. Electron transfer to diazonium ions can be spontaneous or can be controlled electrochemically . To date, a lot of different diazonium salts have been used for graphene modification9.Based on the previous experimental and theoretical experiences with fullerene and carbon nanotubes,  reduction of diazonium ions shows to produce highly reactive free radicals which attack the spThe Gr/Cu (graphene/Cu) interface is extensively studied and is among the candidates for scaling up the production of graphene. In this context the evaluation of the energy level alignment between the organic layer and graphene is of paramount importance to develop hybrid junctions. This feature makes the overall system interesting to develop devices based on a metal/Gr/organic layer interface where the covalently bonded diazonium-derived molecules can interact with the environment and transfer charges to the graphene layer beneath.It is important to note that the covalent attachment of aryl groups onto the graphene surface requires the use of an electrolyte solution, containing acids and salts, that makes this technique potentially exposed to contaminations that could in principle affect the properties of graphene. In order to assess the consequences of electrochemically assisted grafting on the graphene electronic structure, a systematic study of the electronic properties across the different stages of sample preparation and grafting is mandatory.in situ generated 3,4,5-trimethoxybenzenediazonium (TMeOD) cations. This compound is expected to form aryl radicals via electrochemical reduction and to graft the graphene as 3,4,5-trimethoxyphenyl (TMeOP) units10  cation, leading to the grafting of 3,5-bis-tert-butylphenyl (TBP) units, as well as from grafted Highly Oriented Pyrolytic Graphite (HOPG). TBD has been chosen as it does not contain oxygen, allowing for an unambiguous discussion of the oxygen contribution to the measured spectra. Indeed, oxygen may have a three-fold origin, i.e. from the growth solution (water and NO2), from unreacted TMeOP, but also from the Gr/Cu interface. Finally, the band structure at the Γ and K points of the Brillouin zone has been experimentally probed by angle-resolved photoemission spectroscopy (ARPES) with synchrotron radiation. Supported by density functional calculations on 3,4,5-trimethoxybenzene, as well as on TMeOP-grafted on free-standing graphene the analysis of the density of states in the valence band region has also allowed us to identify the HOMO band of TMeOD and set its energy with respect to the Fermi level, thus obtaining a scheme for the band alignment at the Gr/TMeOP hybrid interface.The chemically modified graphene has been characterized through Raman spectroscopy. Then a detailed study of both core levels and valence band has been carried out by comparing the photoemission spectra collected from the pristine, from the treated and from TMeOP-grafted graphene. In order to single out the contribution of the grafted molecular units to the core-level photoemission spectra, the data of the C 1 s, O 1 s, and N 1 s core levels have been contrasted and compared to those collected from graphene exposed to −1, is associated with in plane C-C stretching of sp2 hybridized carbon atoms in the planar graphene skeleton15. The 2D peak is associated with in plane breathing mode of carbon atoms and is a second order Raman scattering process  groups of the TMeOP units are substituted by C4H9 groups where the oxygen is missing, allowing to better single out the different contributions in the carbon and oxygen core levels.After the grafting procedure, core level photoemission spectroscopy measurements were carried out using X-ray photoelectron spectroscopy (XPS) (a) on the pristine graphene on Cu foil (Gr/Cu), (b) on the treated Gr/Cu foil, and (c) on Gr/Cu foil grafted with TMeOP units. XPS spectra have been collected also on (d) 5 mM of TBP-grafted graphene on Cu foil for comparison. The TBD units has been chosen because, as shown in Supplementary Information, the methoxy  and they can be ultimately found anchored to the low sp2 defects of graphene lattice created by the electrochemical process or they can intercalate between graphene and copper.The spectrum of the N 1 s core level can be interpolated by two Lorentzian peaks ascribed to the amine nitrogen group (N-H) at 399.5 eV2 bonding24.The C 1 s spectra are shown in Fig. 9).In addition, other structures appear at larger BE for the treated sample. Two features can be ascribed to the graphene π−π* satellite and to the oxygen contamination (O-C=O contribution3 feature) in the C 1 s confirms the success of the EC grafting procedure and suggests that the graphene coverage by TMeOP molecules is very high. We estimate, as shown in the supplementary, a coverage of about 1 molecule per 3 graphene unit cells. This result is in agreement with the increase of the D peak in the Raman spectrum if we consider the high density defect regime25 ascribed to the grafted molecules. In this approximation, we obtained, in fact, LD=1.07 nm which corresponds to a molecular density of 1 molecule per 4.5 ± 0.5 graphene unit cells in agreement with the coverage estimated by XPS measurements.For the TMeOP-grafted Gr/Cu foil, the presence of the methoxy contribution  are related to the graphene surface contamination due to the air exposure and it is generally ascribed to C-O and H2O groups. The electrochemical process seems to mainly eliminate these oxygen contamination components. This result is in agreement with the literature27, where the electrochemical process is usually adopted to reduce the graphene oxide. Traces of this contamination persist in the treated and grafted samples as proved by the presence of the O-C=O feature.The analysis of oxygen is more complicated Fig. . The eff30.The contribution, at lower binding energy (at about 530.5 eV) in all the samples can be ascribed to the oxygen trapped between the graphene and the copper substrateTo confirm this interpretation, we have verified that when the XPS measurement is more surface sensitive, by changing the analyzer take-off angle from the normal emission  to the take-off angle of 30° (more surface sensitive), the contribution of the oxygen contamination doubles  groups. As shown in the supplementary information  for pristine Gr/Cu and TMeOP-grafted on Gr/Cu at different molecular concentrations Fig. . In the  eV Fig. . When gr eV Fig. . The lac eV Fig. . It is kThe ARPES spectra on the 5 mM TMeOP-grafted Gr/Cu foil Fig.  at the ΓIn order to understand the effects of TMeOP grafting on graphene, we carried out a set of several ab-initio simulations. At first, we considered the isolated 3,4,5-trimethoxybenzene (TMeOB), as shown in Fig. 2.Then, free-standing graphene and TMeOP-grafted graphene, have been considered Fig. . In thes3 C atom and its three C neighbours are lifted of about 0.581 Å and 0.174 Å, respectively. Figure The grafting induces structural and electronic modifications. The graphene is strongly buckled with respect to the unrelaxed, free-standing, graphene plane the sp2 to sp3 at the grafting site. In fact, consistent with literature35, a change in carbon hybridization from sp2 to sp3 in the graphene layer introduces a flat band at the Fermi level, yielding a small peak in the DOS. This state is not present in ARPES measurements. We ascribe this discrepancy to disorder effects that are not accounted for in the supercell calculations. The DFT calculations are in fact carried out considering an ordered structure formed by a TMeOP units bound over a 5 × 5 graphene supercell, as shown in Fig. We note that the electronic structure calculations predict a weak peak (B) in the density of states (DOS) at the Fermi level. The origin of this in-gap state is mainly due to the breakdown of the lattice symmetry of graphene when grafted by TMeOP units and to the change in carbon hybridization from spx/Cu. The other DOS feature ascribable to graphene, peak A in Fig. In spite of the electronic hybridization between the molecule and graphene, it is possible to easily pinpoint the TMeOB contribution in the integral density of states (DOS) spectra, shown in Fig. The interface chemistry of graphene grown on copper and functionalized by covalent bonding with TMeOP units has been studied by core level and angle-resolved valence band photoemission. Two main effects can be ascribed to the electrochemical treatment, the application of the electric field creates a low density of defects in graphene that can accommodate N-groups, in addition the treatment eliminates the oxygen contamination on the top of the graphene layer.x layer between graphene and copper allows to regard graphene as almost decoupled from the metallic substrate and to measure the alignment between graphene σ and π bands and the HOMO of TMeOP, which is found to lay about 1 eV above the top of the σ bands, consistently with DFT calculations.Moreover, comparing the core level spectra collected on both TMeOP and TBP-grafted on graphene, the contribution of the methoxy groups has been singled out in C 1 s and O 1 s spectra. An extra oxygen contribution coming from a CuOTherefore, despite the presence of the solution in the electrochemical cell, that makes this treatment a grafting procedure potentially exposed to contaminations from electrolytes, the results reported in this study show that the functionalization by diazonium chemistry does not affect significantly the chemical environment, and that the electronic properties at the interface are mainly determined by the covalently grafted molecules.36, therefore disclosing the possibility to apply photoemission techniques to systems prepared on the basis of similar electrochemical methods37, and ultimately enabling a scalable pathway for the production of functionalized graphene layers.This finding demonstrates that high quality grafting can be achieved in a set-up with growth conditions less demanding with respect to UHV-related methods2, Pt wire counter and Ag/AgCl/3 M NaCl reference electrodes. 3,5-bis-tert-butylbenzenediazonium (TBD) chloride and 3,4,5-trimethoxybenzenediazonium (TMeOD) chloride are unstable and decompose rapidly: hence they were synthesized from the corresponding aniline precursor immediately prior to electrochemical reduction. This procedure involves 5 mL of a 1 mM (or 5 mM) 3,4,5-trimethoxyaniline  or 3,5-bis-tert-butylaniline  in 50 mM HCl (Sigma-Aldrich) aqueous solution which was mixed with 50 µL (or 250 µL) of aqueous NaNO2 (0.1 M) for activation of the diazotization reaction. Within 90 seconds, this mixture was gently shaken and pipetted into the EC cell. Cyclic voltammetry was used for the electrochemical activation. In the measurements the potential window was chosen from 0.3 V to −0.6 V, scan rate 0.1 V/s for 3 cycles. After modification, the TMeOP modified samples were rinsed with Milli-Q water  to remove any physisorbed material from the surface and dried in a stream of Argon. The STM measurements were acquired with a molecular imaging STM system operating in constant current mode. The tip was obtained by a Pt-Ir wire .Electrochemical measurements were performed using an Autolab PGSTAT101 potentiostat . The electrochemical modification procedure was carried out in a homemade single-compartment three–electrode cell with a working electrode area of 38.5 mmThe substrates used are CVD graphene on Cu foil obtained from Graphenea, graphene on Cu(111) obtained from IMEC and HOPG .38 VG-SCIENTA R3000 analyzer and the Al Kα line of a twin anode X-ray source, operating in ultra-high vacuum conditions (base pressure 2×10−10 torr).In order to avoid the detachment of the phenyl units, all the X-ray photoemission measurements have been collected after an annealing treatment in ultra-high vacuum conditions at temperatures lower than 393 K. Core level spectra have been collected with a properly calibrated39. The overall energy and angular resolution were set to 100 meV and 0.3°, respectively. All the ARPES maps were collected at room temperature (RT) after an annealing treatment. The annealing temperature , in this case, is the minimum value that allows to observe a clear ARPES map.Angle-resolved valence band spectra were carried out at the BaDElPh beamline of the Elettra synchrotron in Trieste  using a photon energy of 34 eV and an hemispherical electron analyzer SPECS Phoibos 150 with a 2D-CCD detector system2. Raman scattering was collected with the same objective and directed to a Raman spectrograph  equipped with a cooled-charge coupled device (CCD) camera operated at −100 °C  through a pinhole, a dichroic mirror  and long pass filter . Accumulation time for each point in ‘grating’ measurement was 1 s. All of the measurements were carried out under ambient conditions and at room temperature.Raman measurements were performed with an OmegaScope 1000 (AIST-NT). Laser light (632.8 nm) from a He-Ne laser was focused onto the sample surface from the side (with an angle of 28° to sample surface) and top, for ‘grating’ and ‘normal’ measurements, respectively, through an objective . Optical density at sample surface was about 500 kW/cm40. Van der Waals corrections to the exchange-correlation potential have been neglected, due to the direct chemical bond induced by grafting. Calculations have been performed with the ABINIT package41 in the framework of Projector-Augmented wave atomic description. A large cut-off energy (700 eV) has been considered for the plane-wave basis definition. All atomic position have been relaxed up to a maximal interatomic force of 10–5 Ha/Bohr. For the graphene and the grafted graphene cells a 7 ×7 ×1 Monkhorts-Pack grid has been adopted, with an adequately large cell size on the out-of-plane direction (15 Å); for the isolated TMeOB, the molecule was placed in a wide (15 Å) cubic cell size, with a single k point. For each case, the convergence on the total energy was set to the 10−9 Ha level.The TMeOB, TMeOD-grafted graphene and pristine graphene electronic structures have been evaluated through ab-initio density functional theory calculations, in the framework of the GGA-PBE approximationSupplementary information.


Sarcoidosis is a systemic granulomatous disease of unknown etiology. Clinical cohort studies of different populations are important to understand the high variability in clinical presentation and disease course of sarcoidosis. The aim of the study is to evaluate clinical characteristics, including organ involvement, pulmonary function tests, and laboratory parameters, in a sarcoidosis cohort at the University of Minnesota. We compare the organ system involvement of this cohort with other available cohorts.We conducted a retrospective data collection and analysis of 187 subjects with biopsy-proven sarcoidosis seen at a tertiary center. Organ system involvement was determined using the WASOG sarcoidosis organ assessment instrument. Clinical phenotype groups were classified using the Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis criteria.Mean subject age at diagnosis was 45.8 ± 12.4, with a higher proportion of males (55.1%), and a higher proportion of blacks (17.1%) compared to the racial distribution of Minnesota residents (5.95%). The majority (71.1%) of subjects required anti-inflammatory therapy for at least 1 month. Compared to the A Case Control Etiologic Study of Sarcoidosis cohort, there was a higher frequency of extra-thoracic lymph node (34.2% vs. 15.2%), eye (20.9% vs. 11.8%), liver (17.6% vs. 11.5%), spleen (20.9% vs. 6.7%), musculoskeletal (9.6% vs. 0.5%), and cardiac (10.7% vs. 2.3%) involvement in our cohort. A multisystem disease with at least five different organs involved was identified in 13.4% of subjects. A restrictive physiological pattern was observed in 21.6% of subjects, followed by an obstructive pattern in 17.3% and mixed obstructive and restrictive pattern in 2.2%. Almost half (49.2%) were Scadding stages II/III. Commonly employed disease activity markers, including soluble interleukin-2 receptor and angiotensin-converting enzyme, did not differ between treated and untreated groups.This cohort features a relatively high frequency of high-risk sarcoidosis phenotypes including cardiac and multiorgan disease. Commonly-utilized serum biomarkers do not identify subpopulations that require or do better with treatment. Findings from this study further highlight the high-variability nature of sarcoidosis and the need for a more reliable biomarker to predict and measure disease severity and outcomes for better clinical management of sarcoidosis patients. Sarcoidosis is a multi-system disease of unknown etiology characterized by the presence of non-caseating granulomas in involved organs . AlthougRecently, four high-risk sarcoidosis manifestations were recognized as highly associated with greater morbidity and mortality . These iThere is significant geographic variability in sarcoidosis manifestations. The variability in sarcoidosis manifestations could be explained by regional variability in sarcoidosis but also could be due to the tools employed for assessment of organ involvement. A number of reports have used the tool developed by the A Case Control Etiologic Study of Sarcoidosis (ACCESS) investigators  to assesOur objective is to describe the clinical characteristics of the sarcoidosis cases seen at the University of Minnesota, and to compare the characteristics of our cohort to the ACCESS and other cohorts. The racial distribution of Minnesota residents includes White (83.75%), Black or African American (5.95%), Asian (4.66%), two or more races (2.81%), other race (1.74%), Native American (1.05%), and Native Hawaiian or Pacific Islander (0.04%) . A uniquThis cohort includes 187 sarcoidosis subjects consented between March 2015 and May 2019 at a tertiary sarcoidosis referral center in Minnesota. The diagnosis of sarcoidosis was made per American Thoracic Society (ATS) statement ) in this cohort (Table n = 37 [19.8%]), including 4.3% with Stage I who were treated, 14.4% with Stage 0 treated, and 1.1% with Stage 0 untreated. Some of the cases who met criteria for Group 1 (multiorgan disease) or Group 9 (cardiac defining therapy) were not included for the respective groups if they met criteria for remitting disease. For instance, while 25 subjects had multiorgan involvement, only 16 of them were classified into Group 1 Multiorgan since six of them were classified into Group 8 Remitting and three of them were classified into Group 9 Cardiac defining therapy. Likewise, while 20 subjects had cardiac involvement, only seven of them who were treated for cardiac involvement and not remitting were classified into Group 9 Cardiac defining therapy.Clinical phenotypes were determined using the GRADS study criteria. Cases with remitting disease, i.e., subjects who were never treated or were off treatment for greater than 12 months, were the largest group (rt Table . Of thosp = 0.003), and lower albumin (p = 0.04) was seen in cases who were treated. White cases had lower IgG (p = 0.01), higher albumin (p = 0.01), and lower total protein levels (p = 0.002) compared to blacks. No significant difference between treated and untreated groups, and between whites and blacks was found for ACE, percent lymphocytes, soluble IL2 receptor, CRP, total bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, calcium, parathyroid hormone, 25-OH Vitamin D, and 1,25 Di OH Vitamin D.The sarcoidosis relevant laboratory studies for this cohort are shown in Table Our cohort has similarities and crucial differences compared to previously described cohorts Table . This coPulmonary sarcoidosis continues to result in significant morbidity and mortality with rising death rates , hospitaA recent NHLBI workshop recognized cardiac involvement as a high-risk phenotype , 32. TheExtra-pulmonary involvement is presumed to be more common in blacks with sarcoidosis. However despite being a cohort composing of predominantly white cases, our cohort had frequent multisystem disease . We observed more frequent involvement of the skin, extra thoracic lymph nodes, liver, spleen, neurologic, cardiac and bone and joint disease. Of these, neurologic, cardiac, and multisystem disease were considered high-risk manifestations of sarcoidosis due to higher morbidity and mortality. Bone and joint involvement is also a frequent cause of impaired quality of life and often requires systemic anti-inflammatory therapy. We included cases with Lofgren’s syndrome in this category due to our observation of significant morbidity during the acute presentation related to arthralgias, although most of these cases had improvement in symptoms and did not need chronic therapy. As the size of our cohort grows, it will be ideally suited for future studies of high-risk sarcoidosis phenotypes.We also examined laboratory studies obtained at the clinic visits. Routine laboratory tests were performed for surveillance of drug-related side effects to assess the disease activity with biomarkers and for asymptomatic organ involvement . We idenWe observed a high proportion of cases that needed systemic anti-inflammatory therapy. Oral corticosteroids are the first-line agents with methotrexate and Imuran as the preferred steroid sparing agent. The dosing of TNF blockers used in sarcoidosis at our center is different than the typical dose for rheumatologic conditions with a maintenance dose of every four weeks (5 mg/kg) for infliximab, in line with the current experience-based recommendations .Despite the retrospective nature of our study, this cohort description provides new insights into regional variability of sarcoidosis as it represents the clinical manifestation in residents of the upper Midwest. Our cohort is a mixed population of incident and prevalent cases and might be a more appropriate representation of sarcoid-related morbidity as opposed to a cohort of newly diagnosed cases. For assessment of pulmonary function testing, we used percent predicted values to assess lung function abnormalities. The other option of using percentile cutoff might be more appropriate, but we do not have this information on all the participants in our study. We also acknowledge that not all the cases in our cohort had all the data points that we presented. As our cohort size grows, we expect the missing values to have a lesser impact on the study findings.Among sarcoidosis cases in our predominantly white Minnesota population, we found a relatively high proportion of advanced and multisystem disease. Lofgren’s syndrome was not common in our cohort despite high percentage of presumed north-European ancestry in our population. Stage II/III pulmonary disease, cardiac disease, multisystem diseases and joint and bone diseases are more frequently detected in this cohort. The analysis of our cohort highlights the lack of utility of serum biomarkers and limitations of pulmonary function tests in assessing prognosis, need for treatment, and disease severity. The higher incidence of cardiac disease, compared to previous cohorts, suggests that cardiac involvement may be under-recognized and highlights the importance of incorporating advanced imaging and a multidisciplinary approach into the evaluation of patients suspected of cardiac involvement. Our cohort is well suited to investigate high-risk sarcoidosis phenotypes, and studying regional variations in disease phenotypes may shed light on disease mechanisms. Incorporating patient reported outcomes in routine care could provide additional insights into indications and outcomes of treatment.