In this work, we applied the spectral element method (SEM) to analyze the dynamic characteristics of fluid conveying single-walled carbon nanotubes (SWCNTs). First, the dynamic equations for fluid conveying SWCNTs were deduced based on the nonlocal Timoshenko beam theory. Then, the spectral element formulation was established for a free/forced vibration analysis of fluid conveying SWCNTs by introducing discrete Fourier transform. Furthermore, the proposed method was validated using several comparison examples. Finally, the natural frequencies and dynamic responses of a simply-supported fluid conveying SWCNTs were calculated by the SEM, considering different internal fluid velocities and small-scale parameters (SSPs). The effects of fluid velocity and SSPs on the dynamic characteristics of SWCNTs conveying fluid were revealed by the numerical results. Compared with other methods, the SEM shows high accuracy and efficiency. Carbon nanotubes (CNTs) have been extensively used in numerous areas due to their excellent mechanical properties ,2. A deeWhen conveying fluid, CNTs have potential applications, such as medical conduits, sensors, and material processing . In thisIn this paper, based on the spectral element method (SEM), which was originally proposed by Doyle  for the V is the velocity of the internal flow and y is the transverse deflection of the tube, i.e., the function of axial coordinate x and time t.The fluid conveying SWCNT with a free body diagram is shown in E is Young’s modulus, G is the shear modulus, I is the moment of inertia of the beam cross section about its neutral axis, k0 is the shear coefficient in Timoshenko beam theory, μ is the Poisson ratio, pA is the area of the beam cross section, pJ is the mass moment of inertia, pm is the mass of tube per unit length, fm is the mass of fluid per unit length, and e0a is the scale coefficient to reflect a small-scale effect {Q=k0c length ,19), Q iQ and M:According to Equations (1) and (2), the following partial differential equation can be obtained after eliminating L is the length of the CNT.By introducing the following non-dimensional parameters, the aforementioned equations will be relevantly simplified:Substituting the aforementioned terms in Equation (5) into Equations (3) and (4), one can obtain the dimensionless equations as follows:η and φ back into Equations (6) and (7), the partial differential equations in the time domain can be transformed into ordinary differential equations in the frequency domain:rη, ω is the dimensionless form of frequency Ω, and the subscript in nω presents the discrete dimensionless frequency. N denotes the number of samples adopted in the discrete Fourier transform, which is a kind of series expansion method [N needs to satisfy N = 2n and n is a positive integer, rτ is the discrete dimensionless time, and i is an imaginary unit.Taking the pairs of the discrete Fourier transform (Equation (8)) of n method . In genen can be omitted, and Equations (6) and (7) becomeSubstituting Equation (8) into Equations (6) and (7), one can get the ordinary differential equations with respect to k is the dimensionless wave number, and C, D are undetermined coefficients.The solution of the ordinary differential Equations (9) and (10) with constant coefficients holds the following exponential form:Substituting Equation (11) into Equations (9) and (10), the following matrix equation can be obtained:The dispersion relation of elastic wave propagation in SWCNT conveying fluid can be obtained using the nontrivial solution condition of Equation (12), i.e.,The four roots of polynomial Equation (13) represent four dimensionless wave numbers. In general, half of them are real roots representing the propagating wave motion, and the other half are imaginary roots corresponding to evanescent wave motion. For one-dimensional wave propagation, one may use Similarly, both the dimensionless shear force and bending moment can be expressed asNow, all the related parameters in the frequency-domain formulations are obtained. In the following section, the spectral formulation of a finite length SWCNT conveying fluid will be established.l, Equation (14) holds the following form if node 1 was chosen as origin [The one-dimensional wave motion in SWCNT  includess origin :The nodal displacement a of the Putting a specific position parameter into Equations (18) and (19) can be formulated in a matrix form, i.e.,Substituting the coefficient column in Equation (20) into Equation (18) yields another expression of nodal displacements as follows:In fact, Equation (22) presents the shape function of the spectral element. Similarly, the nodal force shown in In the same way, the nodal force can be written in a matrix form as follows:Based on Equations (20) and (24), the relationship between the nodal force and nodal displacement can be obtained, i.e.,The natural frequency of the system can be obtained when Equation (26) has nontrivial solution, i.e., For a simply supported SWCNT, both ends are chosen as the element nodes, and the boundary conditions are as follows:Substituting the aforementioned boundary conditions into Equation (26) and picking the related four elements in the matrix, one can get the following homogeneous equation:i th row and j th column of matrix In Equation (28), α are listed in To validate the SEM, we calculated the first five natural frequency parameters eference . The park0 can be obtained by There are several methods for computing the shear coefficient ,49. In t section ,51.It can be seen in α. It can be seen that the natural frequencies decrease with increasing SSP. This means that the SWCNT becomes more flexible accordingly.α; the comparison results are shown in We also computed the first five natural frequency parameters α are shown in α on the natural frequencies is slightly different from that on a simply supported SWCNT. It can be seen in α, except the first-order natural frequency, which actually increases.The plots of frequency versus the log of determinant 3. For the purpose of investigating the effects of α and fluid velocity u on the natural frequencies, we calculated the first five natural frequencies of SWCNT conveying fluid in two cases, i.e., constant fluid velocity u = 0.4 with different SSPs α and constant α = 0.2 with different fluid velocities. The results of the former case are listed in In this example, we analyzed the free vibrations of a simply supported SWCNT conveying fluid with the same material parameters as those in α on the SWCNT conveying fluid is the same as that on the hollow SWCNT. All the natural frequencies decrease with increasing α.As shown in The effect of the internal fluid velocity on the natural frequencies of SWCNT conveying fluid, as shown in β = 0.64, α = 0.3 to compare with that in reference [cu corresponding to zero fundamental frequency is cu = 2.254, which is slightly lower than that predicted by Wang [cu = 2.286. Moreover, the critical velocity of the simply supported fluid conveying SWCNTs was computed with the mass ratio eference . The var by Wang  based onw denotes the wave motion, while the subscripts l and r denote the left and right part of the load position or wave motion, respectively.A simply supported SWCNT conveying fluid, as shown in The SWCNT is divided into two elements when an external point load exists at the middle node p. Accordingly, the spectral matrix in Equation (26) is rewritten in the following global form:The nodal force and displacement column vectors are changed into the following form:For simplicity, the sequence of nodal force and displacement column vectors were rearranged as the following formulas :(32)FR′F(e)The simply supported boundary conditions b and Tf :(34)wB=TNow, the reordered nodal force and displacement vectors in Equation (22) can be rewritten asSubstituting Equations (33) and (35) into Equation (26) and multiplying both sides by With Multiplying both sides of Equation (36) by TTf=02×2 , one canThe unknown nodal displacement vector can be resolved from Equation (37) as follows :(38)w¯R′Once the nodal displacement is obtained, the displacement at any location can be obtained according to the shape function expression of Equation (22):Equation (39) is the dynamic response of the SWCNT conveying fluid in the frequency domain, and that in the time domain can be quickly obtained through the inverse discrete Fourier transform of Equation (39).We also provided an example to demonstrate the validity of the SEM in dynamic response prediction. Up to now, no example of microbeams has been given; as such, the macro-Timoshenko beam was chosen here for comparison . The beax = L/2 are shown in The steady harmonic load is In reference , the timu = 0.4 and α = 0.2 are considered. We finally studied the dynamic response of a simply supported SWCNT conveying fluid under non-dimensional harmonic load u = 0.4, the dimensionless transverse displacement at the center of the tube is drawn in α values.When α increases. This effect of α on the SWCNT vibrations is the same as that in α increases. As seen in α = 0.2, the time history of the dimensionless displacements is shown in When In The SEM for the analysis of flow-induced vibration of a SWCNT conveying fluid was developed based on the nonlocal Timoshenko beam theory. The spectral formulations for free/forced vibration of SWCNT conveying fluid were established. Numerical examples were given to show the validity of the modified method. Some conclusions were drawn, as follows.First, by calculating the first five natural frequencies for a SWCNT and the dynamic response for a macro-Timoshenko beam, the SEM is proved to be effective to predicts the dynamic characteristics of a SWCNT.α on the SWCNT conveying fluid is the same as that on the hollow SWCNT. All the natural frequencies decrease with increasing α.Second, the effect of Third, the effect of the internal fluid velocity on the natural frequencies of SWCNT conveying fluid is the same as that of a macropipe conveying fluid. The flowing fluid weakens the tube effective stiffness and leads to a decrease in the natural frequencies. When the flow velocity approaches the critical value, the first natural frequency drops to zero, and divergence (buckling) instability occurs.Finally, compared with the BEM, the SEM can provide results with the same level of accuracy with a much larger time step, e.g., the time step in the present method is ~500 times higher than that in the BEM.


Vibrational behaviour of symmetric angle-ply layered circular cylindrical shell filled with quiescent fluid is presented. The equations of motion of cylindrical shell in terms of stress and moment resultants are derived from the first order shear deformation theory. Irrotational of inviscid fluid are expressed as the wave equation. These two equations are coupled. Strain-displacement relations and stress-strain relations are adopted into the equations of motion to obtain the differential equations with displacements and rotational functions. A system of ordinary differential equation is obtained in one variable by assuming the functions in separable form. Spline of order three is applied to approximate the displacement and rotational functions, together with boundary conditions, to get a generalised eigenvalue problem. The eigenvalue problem is solved for eigen frequency parameter and associate eigenvectors of spline coefficients. The study of frequency parameters are analysed using the parameters the thickness ratio, length ratio, angle-ply, properties of material and number of layers under different boundary conditions. Composite materials are widely used in petroleum, chemical industry etc., due to the high stiffness and strength. Other characteristics of composite materials are resistance to corrosion and light in weight. The applications of these kinds of materials can be seen in industries like transportation, aircraft, construction, marine and consumer products. Hence, the investigation on the vibrational behaviour of the structure is carried out in order to determine its natural frequency. The important of finding frequency is to prevent the structure from resonance thus to improve the life-span of the structure. There are many literature studied the vibrational behaviour of the shell structure especially in cylindrical shell itself by considering different theories and method of solution. Warburton and Higgs  used RayAmong the researchers those who used Finite Element Method (FEM) in their analysis were Sivadas and Ganesan , Lam andMoreover, the structure can also interact with fluid either the structure filled with, partially with, or submerged. The fluid can be flowing or non-flowing fluid. This type of investigation is known as fluid structure interaction. The characteristics of fluid itself affect the behaviour of the structure. Hence, the investigation has to consider both shell and fluid in order to get better results. Recently, the investigation on fluid structure interaction has get attention among the researchers.Finite Element Method (FEM) was applied in the problem free vibration of isotropic, vertical cylindrical shell partially and completely filled with stationary liquid . The sheGunawan et al.  conducteA study on the vibration of vertical circular cylindrical shell partially filled by an incompressible, compressible, quiescent and inviscid fluid using Rayleigh-Ritz method was analysed by Amabili . The sheVibrational analysis of fluid filled double-walled carbon nanotubes using the wave propagation method was carried out by Natsuki et al.  and the A dynamic stiffness method was studied by Tran and Manh  to invesAccording to a nonlocal theory, there are two types of models which are structural hardening and softening models. Li et al.  and ShenDifferent approach study is done in order to study the fluid structure interaction. The aim of this study is to determine the vibrational behaviour of symmetric angle-ply laminated composite cylindrical shell. The quiescent fluid-filled shell is considered. The quiescent fluid means that the vibration of fluid is depends on the vibration of the shell. Once the shell vibrates, the fluid will vibrate accordingly. Bickley-type spline is used to approximate the displacements and rotational functions since it gives better accuracy and also uses of lower order approximation in solving boundary value problem . The shex,θ,z) where x is along the axial direction, θ is in the circumferential and z is along the normal direction. The length of the shell is h and radius is r. The displacement components u,v,w are expressed under the first order shear deformation theory  is divided. These splines are lower order approximation and an effective method since it has fast convergence and high accuracy. The points of division N+5 homogeneous equations into 5N+15 unknown spline coefficients ai,bj,ci,dj,ei,fj,gi,pj,li,qj  is obtained.Here, en by Eq  are satien by Eq  at the kTwo types of boundary conditions are used to analyse the problemClamped-Clamped (C-C)(both the ends are clamped);Simply supported–Simply supported (S–S)(both the ends are simply supported);N+5 homogeneous equations, one can get 5N+15 equations in the same number unknown coefficients. This can be written asThe system will get ten more equations on spline coefficients by imposing any one of the boundary conditions. Combining these ten equations with the earlier 5P] and [Q] are the square matrices of order (5N+15)×(5N+15). {q} is the column matrix of the spline coefficients of order (5N+15)×1 and λ is the frequency parameter.Here, [N is taken as 16 since the change in percentage for next following values of N is 0.29%.The convergence study has been made in order to determine the number of iteration. This is done by fixing the circumferential node number, thickness ratio, length ratio, number of layers and orientation of the material under C-C and S-S boundary conditions. The number of knots, ωm of three layered symmetric angle-ply shells using the materials Kevler-49 epoxy (KGE) and AS4/3501-6 Graphite/epoxy (AGE) are arranged in the order of KGE-AGE-KGE with the angle orientations at (30°/0°/30°) are presented. The parameters n = 2, H = 0.02 are fixed. The shell is clamped at both the ends. According to In the case of comparison studies, none of literature has been done on symmetric angle-ply shell with consideration of fluid. There are studies on symmetric angle-ply shells but limited to empty shells only , 13. HenK = 5/6 is fixed throughout this analysis [Three and five layered symmetric angle-ply circular cylindrical shells with fluid are analysed. The materials which are KGE and AGE are used . The sheanalysis  and firsωm on length parameter is studied for three layered shell with the combination of the materials KGE and AGE with C-C boundary conditions. The parameters n = 4 and H = 0.015 are fixed and the ply-angles are arranged as 30°/0°/30°, 45°/0°/45° and 60°/0°/60° using the materials AGE and KGE and ordered as KGE-AGE-KGE. Since the frequency parameter λm is explicitly a function of length of the cylinder, hence when studying the influence of the length of the cylinder on its vibrational behaviour, the angular frequency ωm is considered instead of λm. The relationship between the frequency and the length of the shell can be seen as in [en as in . The naten as in .L ranging from 0.5 to 0.75. Later, it decreases slowly. It also can be seen that the frequency for m = 1 of m = 2,3, the values of the frequencies are the lowest for From the analysis, it can be seen that the frequency decreases as the length parameter increases and the frequency is higher for higher modes. The frequency decreases fast at ωm of five layered angle-ply arranged materials in the order KGE-AGE-KGE-AGE-KGE with ply-angles of 45°/30°/0°/30°/45°, 30°/45°/0°/45°/30° and 60°/30°/0°/30°/60°, respectively for C-C conditions. The values of n = 4 and H = 0.02 is fixed. The figure shows that the frequency decreases as the length parameter increases. The investigation is continued by applying S-S boundary conditions using the same parameters as shown in ωm in ωm in λm by considering three layered shells arranged in KGE-AGE-KGE materials with n = 4 and L = 1.5 are fixed under C-C boundary conditions. The shell is arranged in the order of 30°/0°/30°, 45°/0°/45° and 60°/0°/60°. From the analysis, it can be seen clearly that the frequency rises as the thickness parameter rises. The frequencies are greater for larger modes. The values of λm in λm under S-S boundary conditions. Results show that the values of λm in λm in λm with respect to the thickness parameter for five layered angle-ply by fixing n = 4 and L = 1 for C-C boundary conditions. The materials are arranged as KGE-AGE-KGE-AGE-KGE with angle orientation at 45°/30°/0°/30°/45°, 30°/45°/0°/45°/30° and 60°/30°/0°/30°/60°. Clearly, the frequency is higher for higher values of thickness parameter. λm under S-S boundary conditions are lower compared to the values of λm under C-C boundary conditions.n = 3, the effects of length parameter for both three and five layered shells on frequencies under C-C boundary conditions are investigated as depicted in ωm < 0.75. Also, the frequencies of five layered shell are higher than the frequencies of three layered shell.By fixing Next, the frequencies with respect to thickness parameter are analysed as shown in The vibrational behaviour of layered cylindrical shell with symmetric angle-ply is investigated using spline method. The shell contained a quiescent fluid. The equations of motion of the shell are based on first order shear deformation theory. Investigations on empty and fluid-filled shells show that the frequency value reduces as the fluid term is included. This is due to the fluid in the shell that provides added mass to the shell.Results show that by increasing the length of the shell, the frequency decreases. In contrast, the frequency increases as the shell thickness increases. Meanwhile, frequency of C-C boundary conditions is higher than the frequency of S-S boundary conditions. It can be concluded that the geometric parameters, material properties, angle orientations, number of layers and boundary conditions significantly affects the frequency of the shell.


Target enrichment combined with chromosome conformation capturing methodologies such as capture Hi-C (CHC) can be used to investigate spatial layouts of genomic regions with high resolution and at scalable costs. A common application of CHC is the investigation of regulatory elements that are in contact with promoters, but CHC can be used for a range of other applications. Therefore, probe design for CHC needs to be adapted to experimental needs, but no flexible tool is currently available for this purpose.We present a Java desktop application called GOPHER (Generator Of Probes for capture Hi-C Experiments at high Resolution) that implements three strategies for CHC probe design. GOPHER’s simple approach is similar to the probe design of previous approaches that employ CHC to investigate all promoters, with one probe being placed at each margin of a single digest that overlaps the transcription start site (TSS) of each promoter. GOPHER’s simple-patched approach extends this methodology with a heuristic that improves coverage of viewpoints in which the TSS is located near to one of the boundaries of the digest. GOPHER’s extended approach is intended mainly for focused investigations of smaller gene sets. GOPHER can also be used to design probes for regions other than TSS such as GWAS hits or large blocks of genomic sequence. GOPHER additionally provides a number of features that allow users to visualize and edit viewpoints, and outputs a range of files useful for documentation, ordering probes, and downstream analysis.https://github.com/TheJacksonLaboratory/Gopher.GOPHER is an easy-to-use and robust desktop application for CHC probe design. Source code and a precompiled executable can be downloaded from the GOPHER GitHub page at The online version of this article (doi:10.1186/s12864-018-5376-4) contains supplementary material, which is available to authorized users. Functional elements that are widely separated in the linear sequence of the genome can be brought into contact with one another by the folding of the genome in three-dimensional space. A series of extensions of the original targeted chromosome conformation capture 3C) method that was introduced in 2002  culminatC method CHC has been used in a variety of experimental settings to provide more in-depth data for specific loci than would be feasible with Hi-C. For example, promoter CHC focuses on the enrichment of gene promoters in order to identify functional interactions with distal regulatory elements such as enhancers –10. OtheCHC has also been used to analyze gene regulation programs in differentiation and disease –22 by prCHC employs a set of biotinylated oligonucleotides that are designed to hybridize to and ’capture’ target sequences; such oligonucleotides are usually referred to as baits or probes. Several technologies are commercially available for capture of exonic sequences in exome sequencing . These mTo our knowledge, only two tools are available for capture Hi-C probe design. CapSequm  is a webGenerator Of Probes for capture Hi-C Experiments at high Resolution), an easy-to-use Java-based desktop application that provides a suite of methods and visualization tools for the automated design and subsequent manual curation of viewpoints. GOPHER enables all steps required for probe design to be performed in a unified framework that leads users from the download of the genome, alignability, and transcript files, through the choice of parameters such as target genes or regions, restriction enzymes, and desired thresholds for GC content, alignability, and digest length. Users can inspect the genomic context of each of the generated viewpoints, and can add or remove digests (restriction fragments) if desired. GOPHER implements three main approaches to probe design, including two that have not previously been available. GOPHER outputs a series of files including a probe file that can be used to order probes (baits) for the enrichment of the targeted regions in capture Hi-C experiments. Additionally, summary statistics are generated that can be used for documentation of the final design. Users can generate a digest file containing attributes of the selected and unselected digests relevant for downstream analysis.Here we present GOPHER , and two probes are placed at the outermost ends of the digest. The simple-patched approach “patches” viewpoints that are poorly covered by single digests. GOPHER additionally implements a new approach to probe design that we term extended, which is intended to provide greater resolution than the simple approach by performing restriction digestion with a 4-cutter instead of 6-cutter and selecting sets of multiple fragments per target region. In general, the simple and simple-patched approaches are best suited for investigations of larger numbers of targets such as a promoterome in which all promoters of all coding genes are investigated , 8, 10, In order to design CHC probes, users need to download and preprocess a substantial amount of sequence and annotation data. GOPHER provides a graphical user interface (GUI) to streamline these tasks Fig. a. VariouP53 instead of the official gene symbol TP53), GOPHER will issue a warning and report a list of unmappable symbols that can be used to search for the current correct symbols. An alternative shortcut option allows promoters of all protein coding genes to be selected as targets. GOPHER also accepts a BED file with genomic positions. For instance, the coordinates of GWAS hits can be uploaded in BED6 format.Following this, users specify the desired enrichment targets Fig. b. For prGOPHER allows the user to set a number of parameters that control the choice of viewpoints, digests, and probes Table  using a Capture Hi-C probes must meet certain requirements that are substantially different from the those for standard use cases such as exome sequencing. Note that in this article, we refer to the DNA sequences produced by the sonication step of next-generation sequencing as fragments, and we refer to the DNA sequences produced by restriction digestion as digests. Within Hi-C libraries, interacting sequences are represented by hybrid molecules consisting of two pieces of digests from different genomic locations Fig. a. The sobmin usable probes. There is no restriction on the overlap between probes, because we reasoned that the sequences directly next to the cutting sites occur most likely within hybrid fragments  necessary to select a digest for enrichment. By default, GOPHER demands that each of the two margins of a digest contain bmin probes; if this is the case, the digest is referred to as balanced. If the user allows unbalanced margins in the Setup tab of GOPHER, then any digest with at least 2·bmin valid probes will be selected. If the two margins do not have equal numbers of probes, then the digest is referred to as unbalanced  were targeted with two probes each . For instance, 0.25 means that the corresponding k-mer occurs 4 times within the entire genome. These tracks were created in the course of the ENCODE project  using thp↦ak(p) indicates how often the k-mer starting at position p occurs in the entire genome. For instance, a50(42)=2 means that the 50mer starting at position 42 occurs two times in total. GOPHER uses binary search for efficient retrieval of all k-mer alignabilities within a given target region which can be used for fast calculation of mean k-mer alignabilities of digests. For a probe of length l starting at position p, the mean k-mer alignability is calculated as follows: Within GOPHER, the alignability map By default, probes with a mean k-mer alignability above 2 are rejected, but users can adjust this threshold value.GOPHER uses the HTSJDK library to retrieve the sequences of candidate digests and probes and counts the number of G, C, A, and T bases in order to calculate the GC content. By default, probes with a GC content of between 35 and 65% are accepted, but users can adjust these threshold values.μ=0 that corresponds to the TSS and a standard deviation Ideally, the selected digest of a simple viewpoint is centered at the TSS and not too short. In order to capture this intuition, we estimate the average size max,TSS+downmax]. For a good viewpoint, the selected digests should cover as many positions as possible within the specified range, especially near the TSS.The score for extended viewpoints is very similar to the score for simple viewpoints but it allows asymmetric length ratios for the targeted regions upstream and downstream of the TSS. Given the user-specified maximum allowed upstream and downstream distances from the TSS Table , an exteTo calculate the empirical score, we model the coverage of a viewpoint and its associated upstream and downstream sizes (5000 bp and 1500 bp by default) as based on normal distributions with mean at the transcription start site  and standard deviations calculated as 1/6 times the upstream or downstream size. Since 6 standard deviations cover ∼ 99.999% of the total probability, if chosen digests cover all of the upstream space they will add 0.5 to the total probability and similarly with the downstream digests. In general, a digest will contribute more to the total probability the closer it is to the TSS. The probability is multiplied by 100 and the score is reported as a percentage.Project name: GOPHER: Generator Of Probes for capture Hi-C Experiments at high ResolutionProject home page:https://github.com/TheJacksonLaboratory/GopherOperating system: Platform independentProgramming language: JavaOther requirements: Java 1.8License: JAXAny restrictions to use by non-academics: License neededAdditional file 1Supplementary figures for the distribution of mapped Hi-C interaction read pairs within digests, an exploration of alignability thresholds, heterogeneous definitions of viewpoints and explanation of GOPHER’s allTracks.bed file. (PDF 1430 kb)


FGFR3 mutations are generally found in a luminal papillary tumour subtype that is characterised by better survival than other molecular subtypes. To better understand the role of FGFR3 in invasive bladder cancer, we examined the process of tumour development induced by the tobacco carcinogen OH‐BBN in genetically engineered models that express mutationally activated FGFR3 S249C or FGFR3 K644E in the urothelium. Both occurrence and progression of OH‐BBN‐driven tumours were increased in the presence of an S249C mutation compared to wild‐type control mice. Interestingly, at an early tumour initiation stage, the acute inflammatory response in OH‐BBN‐treated bladders was suppressed in the presence of an S249C mutation. However, at later stages of tumour progression, increased inflammation was observed in S249C tumours, long after the carcinogen administration had ceased. Early‐phase neutrophil depletion using an anti‐Ly6G monoclonal antibody resulted in an increased neutrophil‐to‐lymphocyte ratio at later stages of pathogenesis, indicative of enhanced tumour pathogenesis, which supports the hypothesis that suppression of acute inflammation could play a causative role. Statistical analyses of correlation showed that while initial bladder phenotypes in morphology and inflammation were FGFR3‐dependent, increased levels of inflammation were associated with tumour progression at the later stage. This study provides a novel insight into the tumour‐promoting effect of FGFR3 mutations via regulation of inflammation at the pre‐tumour stage in the bladder. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.Recent studies of muscle‐invasive bladder cancer show that  The majority of bladder cancers are urothelial cell carcinomas (90%), followed by squamous cell carcinomas. Smoking is a major risk factor for bladder cancer. The majority of urothelial cell carcinomas at diagnosis are non‐muscle‐invasive (NMIBC) (70%), the remainder showing muscle invasion. Muscle‐invasive bladder cancer (MIBC) without metastasis is managed by neoadjuvant chemotherapy followed by radical cystectomy Bladder cancer is the tenth most common cancer type, particularly in ageing men  mutation and overexpression are common in bladder cancer FGFR3 mutation and overexpression are associated with urothelial‐like or luminal papillary tumour subgroups that are characterised by better survival than other molecular subtypes FGFR3C746C > G, encoding the FGFR3 S249C oncoprotein, accounts for 48–71% of all FGFR3 mutations in non‐invasive urothelial cell carcinoma In vitro studies have provided evidence that mutational activation of FGFR3 through S249C or K644E can modestly increase cell proliferation and reduce apoptosis, and that various FGFR inhibitors are effective in its functional suppression FGFR3‐mutated advanced bladder cancer after failure of platinum‐based chemotherapy FGFR3 is one of four tyrosine kinase receptors for FGFs FGFR3 mutations in tumour pathogenesis and progression will help in interpreting trial outcomes and allow further stratifications. The use of in vivo models closely reflecting the disease conditions would increase robustness and confidence in translation of pre‐clinical findings to trials. Previously, we showed in a mouse model of spontaneous tumour formation that murine Fgfr3 K644E  in combination with Pten loss was able to induce morphological changes in the urothelium with cellular characteristics indicative of abnormal differentiation N‐butyl‐N‐(4‐hydroxybutyl)‐nitrosamine (OH‐BBN), derived from tobacco smoke A better understanding of the role of Fgfr3 K644E model In this study, we have generated a novel transgenic mouse line that expresses FGFR3 S249C in the urothelium and have compared the effects of OH‐BBN with a wild‐type control, as well as with the previously reported Tg(UroII‐hFGFR3IIIbS249C) (‘FGFR3S249C’) is described in the supplementary material, Supplementary materials and methods. UroIICre Fgfr3+/K644E mice were generated as previously described Generation of Mice were administered 0.05% v/v OH‐BBN  in drinking water three times a week for 10 weeks, starting from 8 to 16 weeks of age, followed by 10 weeks of water. All experiments were performed according to an approved Project Licence under the Home Office Animal (Scientific Procedures) Act 1986.Wild‐type mice were injected (i.p.) with 500 µg of either 1A8 monoclonal antibody  or 2A3 isotype control  three times per week for 10 weeks, with concurrent OH‐BBN administration in drinking water. Blood was collected in EDTA‐containing tubes by cardiac puncture following euthanasia. White blood cell populations were analysed using a ProCyte Dx Hematology Analyzer .Methods and antibody details are provided in the supplementary material, Supplementary materials and methods.in situ (CIS), tumour); ‘large’ tumour, tumour size more than 50% of the bladder; ‘small’ tumour, tumour size less than 50% of the bladder; invasiveness ; lobulation of the basement membrane ; squamous transformation . Urothelial phenotype at 2 weeks were scored for atypia/dysplasia . Inflammatory phenotype and neutrophils were scored for thickness of the stroma ; angiogenesis in the inner stroma and in the outer stroma . Neutrophil infiltrations at 2 and 12 weeks were scored in the urothelium, or stroma and muscle, using the criteria <5, 6–20, 21–50, and >50, where section size of the bladders were comparable among samples. Inflammatory phenotype at 20 weeks was scored using the criteria modified from Klintrup et al's method The number of mice showing the specific criterion was recorded, as assessed on one cross‐section per bladder. On the rare occasion that multiple lesions with different scoring criteria were present within one section, the more severe criterion was assigned. The criteria used for tumour phenotype were: Stage of pathogenesis  (‘FGFR3S249C’). The histological appearance of the FGFR3S249C urothelia (n = 17) appeared normal at 12 months of age , which we reported earlier FGFR3 mutation by itself is not able to induce urothelial pathogenesis. Furthermore, bladders of double‐mutant mice with both FGFR3S249C and Pten loss, ‘FGFR3S249CPten’, did not show any noticeable histological abnormalities at 12 months of age (n = 12)     which invaded the stroma     Figure D–F. Lobu) Figure E, F. A sFGFR3 K644E mutation, indicating differential regulation of urothelial pathogenesis by the two FGFR3 mutations.Taken together, the increase in tumour pathogenesis became evident as early as 12 weeks from the start of the carcinogen treatment. Unexpectedly, at an early phase of carcinogen induction (2‐week time point), the histopathological changes in the urothelium were suppressed in the presence of the FGFR3 mutant urothelium, we compared the effects of DNA damage caused by OH‐BBN by analysing the levels of γH2AX, p53, p21, and Ki67 , as well as indicators for signalling pathways, including phosphorylation of ERK, AKT, cJUN, and STAT3 (data not shown). No alterations were found in DNA damage response or downstream signalling pathways in the FGFR3S249C urothelium.In an attempt to understand the mechanisms that underlie tumour pathogenesis in FGFR3 mutant and wild‐type cohorts . Recruitment of neutrophils to the urothelium, the stroma, and the muscle layer was clearly observed  . At 12 weeks, neutrophil recruitment was no longer suppressed in FGFR3S249C   , while the other was not (score 0). The level of T‐cell infiltration was similar in FGFR3S249C and wild‐type tumours .We also investigated the status of tumour inflammation at 20 weeks. Interestingly, tumours were mildly more infiltrated by inflammatory cells in FGFR3 S249C mutation at the pre‐tumour stage (2 weeks), while at 20 weeks, FGFR3S249C bladders were mildly more inflamed than wild‐type.These results indicate that the acute inflammatory response to carcinogen treatment, particularly the recruitment of neutrophils to the bladder, was transiently suppressed in the presence of + (1A8) along with OH‐BBN treatment in a cohort of wild‐type mice The early suppression of an acute inflammatory response could result in chronic inflammation later along the process of tumour progression, leading to enhanced tumour pathogenesis in the bladder. To investigate this, neutrophils were depleted using a monoclonal antibody against Ly‐6Gp = 0.0317)  Figure F.FGFR3S249C mice at the pre‐tumour stage could account for increased tumourigenesis.The results of the neutrophil depletion study indicated that the impairment of the acute inflammatory response at the pre‐tumour stage could lead to a later increase in the levels of circulatory immune cells, indicative of enhanced progression of bladder tumours. This supports our hypothesis that the transient suppression of neutrophil recruitment to the bladder in FGFR3 mutant proteins may have regulated the level of inflammation) or, alternatively, phenotype‐dependent (i.e. the inflammatory phenotype may have been caused by the severity of bladder/tumour pathology). In order to address this, we analysed the correlation between genotypes (cohort), phenotypes, and inflammation .Changes in the levels of inflammatory infiltrations could be genotype‐dependent  . The Kruskal–Wallis test showed that differences seen among cohorts were statistically significant in dysplasia (p = 0.025), neutrophils in the stroma (p = 0.003), and in the muscle (p = 0.017) , indicating that both dysplasia and inflammation were genotype‐dependent.At 2 weeks, dysplasia significantly correlated with the cohort  and neutrophils in the stroma  . Neutrophils in the stroma were also correlated with bladder phenotypes, including lobulation , and squamous transformation . However, no statistically significant links were observed regarding neutrophils by the Kruskal–Wallis tests, either controlled by genotype  or by pathogenesis .At 12 weeks, cohort‐dependent differences were evident in lobulation  . Scores of inflammation were notably correlated with those of pathogenesis. The Kruskal–Wallis test showed that inflammation in tumours was associated with pathogenesis  and invasiveness ; however, no association was evident when controlled by cohort .At 20 weeks, a positive correlation was evident in scores that indicate tumour pathogenesis  and inflammation  . A significant correlation with cohort was seen in squamous transformation , inflammation was no longer regulated by FGFR3 mutations. At 20 weeks, only tumour pathogenesis was associated with the FGFR3 genotype, and inflammation was associated with tumour progression.Taken together, at an early phase of carcinogen induction, regulation of pathogenesis and inflammatory response was associated with the FGFR3 mutation are associated with the urothelial‐like or luminal papillary tumour subgroup that is generally characterised by lower levels of lymphocytic infiltration FGFR3 mutation status and compared the immune gene expression signature FGFR3 mutation did not influence the level of immune signature in any of the urothelial‐like/luminal subtypes . When subtypes were grouped together, human urothelial‐like/luminal tumours with FGFR3 mutation were less immune‐infiltrated than those with wild‐type status . However, this is due to the prevalence of urothelial‐like A‐progressed (UroA‐prog)/luminal‐papillary subtypes that harbour FGFR3 mutations more frequently, and these subtypes were less immune‐infiltrated among the group regardless of FGFR3 mutation status .In humans, tumours with FGFR3 mutations using OH‐BBN‐induced, genetically engineered mouse models of invasive bladder cancer, we report three significant findings: firstly, in the presence of mutationally activated FGFR3 S249C, there was an increased number of mice that developed bladder tumours and the tumour phenotype was more advanced . No significant differences were observed in the levels of T‐cells . Neutrophil depletion at an early phase increased the NLR later at 20 weeks, during the timeframe of tumour establishment and progression, indicative of enhanced tumour pathogenesis . This included urothelial‐like B (UroB), a subtype with the worst overall survival, similar to small‐cell/neuroendocrine‐like (Sc/NE‐like) FGFR3 mutation when all subtypes within the urothelial‐like/luminary papillary subtypes were combined; however, this is due to the frequency of lower‐infiltrated subgroups, such as UroA‐Prog/luminal‐papillary subtypes . The lack of a difference in immune gene expression within the tumour subtype in the presence and absence of FGFR3 mutation in human MIBC could be due to the fact that human studies are based on established tumours, while our study in mouse models investigated the functional effects of FGFR3 along the process of tumour initiation and development, the earlier phases in tumour pathogenesis. It would be interesting to compare the levels of tumour inflammation in OH‐BBN‐induced bladder models with other molecular changes, such as Tp53, Stat3, and Sparc models FGFR3 mutations, early suppression of neutrophil infiltration was not present in the Fgfr3K644E mice , Ki67 (D‐F) and anti‐FGFR3 (B‐9) antibody (G‐I). (D‐F) Few cells within the urothelium showed positivity with cell proliferation marker Ki67. Mainly stromal cells were positive. No significant difference in Ki67 staining was observed between Wildtype Control, FGFR3S249C or FGFR3S249CPten mice. (G‐I) Similar levels and patterns of FGFR3 expression were observed in the Wildtype, FGFR3S249C and FGFR3S249CPten urothelium. Immunohistochemistry was performed in n=3 samples per genotype. Scale bar represents 100 µm (A‐I).Figure S2. Histopathology of the urothelium and the bladder tumours at 20 weeks from the start of the carcinogen treatment. The phenotype was analysed in male and female cohorts separately. Frequency of the observed phenotype was shown as percentage within the samples analysed. (A) Pathogenesis observed in the bladder. 23.4% of Wildtype mice (n=11/47) formed well‐recognisable tumours of an invasive nature . When male and female mice were analysed separately, tumours had occurred in 40.0% (n=8/20) and 11.1% (n=3/27), respectively. This is in concordance with the increased frequency of bladder cancer in males. (B) Invasiveness of the urothelial and tumour cells. (C) Lobulated appearance of basement membrane. (D) Squamous differentiation observed in the urothelium and the tumour. Number of samples analysed for each phenotype is indicated below each column. The p‐values (Mann‐Whitney) are indicated above the columns, when significant (* <0.05 and ** < 0.005).Figure S3. Histopathology of the bladder at 2 and 12 weeks from the start of the carcinogen treatment. Phenotype was analysed in both genders (M/F), male and female cohorts at 2 weeks (A) and 12 weeks (B‐D). The frequency of each phenotypic criterion was shown as percentage within mice analysed. Presence of atypia and dysplasia (A) in the urothelium at 2 weeks, (B) Pathogenesis observed in the bladder, (C) lobulated appearance of the basement membrane and (D) squamous differentiation observed in the urothelium at 12 weeks were scored. Results of the analysis of M/F combined for atypia/dysplasia at 2 weeks, pathogenesis and lobulation at 12 weeks are presented in Figure 3. Number of samples analysed is indicated below panels. The p‐values (Mann‐Whitney) are indicated where significant (* <0.05 and ** < 0.005).Figure S4. Response to DNA‐damaging effects of OH‐BBN treatment. The absence of specific differences in the pattern of γH2aX phosphorylation, expression of p53, p21, proliferation between Wildtype and FGFR3S249C, excludes dysregulation of the Phospho‐γH2aX‐p53 axis as the potential cause of malignancy. IHC was performed in the minimum of n=5 samples per cohort at each time point. Representative sections of Wildtype and FGFR3S249C bladders without OH‐BBN treatment , at 2 weeks  and 12 weeks from the start of the OH‐BBN treatment  stained by Phospho‐γH2aX (A‐F), p53 (G‐K), p21 (L‐P) and Ki67 (Q‐V). When untreated with OH‐BBN, FGFR3S249C bladders showed similar staining pattern to controls, including p53 and p21 . In the absence of carcinogen treatment, γH2aX phosphorylation was scarcely seen, indicating that the DNA damage was minimal in both cohorts . Proliferation of the urothelial cells was similarly low in Wildtype and FGFR3S249C , consistent with our earlier observations . At 2 weeks of OH‐BBN treatment, increased γH2aX phosphorylation was seen in Wildtype and FGFR3S249C  compared to untreated urothelia . p53 protein levels were elevated along the basal cell layer as well as in some of the intermediate cells . p21 expression was evident in all urothelial layers . Increased proliferation was typically found along the basal layer of cells . These changes appeared to be according to the expected response to the carcinogen, and were similar between Wildtype and FGFR3S249C cohorts. At 12 weeks, γH2aX phosphorylation generally remained in all layers of the urothelium in Wildtype, as well as in FGFR3S249C mice . However, cells that had undergone squamous cell transformation appeared to have lost γH2aX phosphorylation in the basal cell layer (F), indicating that cells with DNA damage were cleared from these areas. Expression of p53 was strong in all layers of the urothelium, in particular along the basal cell layer and in some of the intermediate cells , while some urothelial regions lacked p53 expression (K). Compared to 2 weeks of OH‐BBN exposure, the expression of p21 was less intense at 12 weeks  and no longer showed any association with regions of high p53 expression as seen at 2 weeks. Proliferation was generally observed along the basal cell layer and often regionally intense . Scale bar represents 100 µm in all panels.Figure S5. Inflammatory characteristics of the bladder at 2 weeks of carcinogen treatment. Phenotype was analysed in both genders (M/F), male and female cohorts and shown as percentage within the samples analysed. (A) Thickness of the stroma and (B) the presence of blood vessels in the stroma closer to the urothelium (inner stroma). No difference was detected in the presence of blood vessels closer to the muscle (the outer stroma) comparing cohorts (data not shown). (C) Macrophages in the urothelium were scored using IHC with F4/80. There were no differences in the number of macrophages in the stroma and muscle comparing Wildtype and FGFR3S249C (data not shown). Presence of neutrophils in the urothelium (D), stroma (E), and muscle layer (F) of the bladder. Number of samples analysed is indicated below panels. Number of samples analysed is indicated below each column. The p‐values (Mann‐Whitney) are indicated where significant (* <0.05).Figure S6. Presence of the neutrophils in the bladder at 12 weeks of carcinogen treatment. Presence of neutrophils in the urothelium (A), stroma (B), and muscle layer (D) of the bladder at 12 weeks from the start of OH‐BBN treatment. Phenotype was analysed in male and female cohorts and shown as percentage within the samples analysed. Number of samples analysed is indicated below each column in C. The p‐values (Mann‐Whitney) are indicated where significant (* <0.05).Figure S7. Inflammatory phenotype of the bladder at 20 weeks from the start of application of carcinogen. Presence of overall levels of inflammatory infiltrations (A) and neutrophils (B) in the tumour was analysed in males and females individually. Infiltration of the T‐cell population was analysed using CD3 as a marker in the urothelium (C), CIS (D) and in tumours (E). The Y axis indicates percentage within the samples analysed. Number of samples analysed is indicated below each column. The p‐values (Mann‐Whitney) are indicated where significant (* <0.05).Figure S8. Presence of neutrophils in the bladder tissues upon neutrophil depletion. (A) Efficiency of neutrophil depletion was evaluated in the bladder tissues at 2 weeks from the start of the carcinogen treatment. (B) Infiltration of neutrophils in the tumour were examined at 20 weeks time point. The Y axis indicates percentage within the samples analysed. Number of samples analysed is indicated below each column. Differences were not statistically significant.FGFR3 mutant and Wildtype stratified by Lund or TCGA molecular subtype.Figure S9. Immune signature expression in the TCGA dataset comparing  Immune infiltration signature in Lund Urothelial‐Like subtype (n=170) (A), TCGA Luminal subtype (n=246) (B), and all tumours grouped by FGFR3 mutation status in Lund Urothelial‐Like subtype (C), and TCGA Luminal subtype (D). The p‐values (Wilcoxon rank test) are indicated. The expression of immune genes was not affected strongly by the presence or absence of FGFR3 mutations within the different subtypes of the Lund or TCGA classification . The immune expression did not vary significantly within Urothelial‐like B (UroB) based on FGFR3 mutational status (p=0.83), while UroB as a whole and UroB with FGFR3 mutations were more infiltrated than Urothelial‐like A‐Progressed (UroA‐Prog) (p=0.0067 and 0.01 respectively) (A). FGFR3 mutations are uncommon in the Urothelial‐like Infiltrated (Uro‐Inf) and Urothelial‐like C (UroC) groups. The infiltration level in Uro‐Inf and UroC was higher than in UroAProg tumors . Similarly, no difference in infiltration was seen between FGFR3 mutated and wild‐type tumors of the TCGA Luminal‐Infiltrated group (p=0.92) (B). TCGA Luminal‐Infiltrated had higher immune infiltration than the Luminal‐Papillary group (p<2.2e‐ 16).  When grouped by FGFR3 mutation status, it appears that FGFR3 mutated Urotheliallike/ Luminal tumors had moderately lower immune infiltration. However, the lower infiltration seen in the mutated cases is due to an enrichment of mutations in the less infiltrated UroA‐prog and Luminal‐Papillary subtypes, since the mutated cases in C, D are not significantly different from FGFR3 wild‐type UroA‐prog/Luminal‐papillary tumours, as shown in A, B.Click here for additional data file.Table S1. Summary of phenotype compared by genderTable S2. Correlation of inflammatory phenotype by cohort and by bladder phenotype at 2 weeksTable S3. Correlation of inflammatory phenotype by cohort and by bladder phenotype at 12 weeksTable S4. Correlation of inflammatory phenotype by cohort and by tumour phenotype at 20 weeksClick here for additional data file.


Twitter is growing in popularity and influence among emergency physicians (EP), with over 2200 self-identified EP users. As Twitter’s popularity has increased among EPs so too has its influence. While there has been debate about the value of Twitter as an effective educational delivery tool, little attention has been paid to the nature of the conversation occurring on Twitter. We aim to describe how influential EPs use Twitter by characterizing the language, purpose, frequencies, content, and degree of engagement of their tweets.We performed a mixed-methods analysis following a combined content analysis approach. We conducted qualitative and quantitative analyses of a sample of tweets from the 61 most influential EPs on Twitter. We present descriptive tweet characteristics and noteworthy themes.We analyzed 1375 unique tweets from 57 unique users, representing 93% of the influential Twitter EPs. A majority of tweets  elicited some response in the form of retweets, likes, or replies, demonstrating community engagement. The qualitative analysis identified 15 distinct categories of tweets.Influential EPs on Twitter were engaged in largely medical conversations in which most messages generated some form of interaction. They shared resources and opinions while also building social rapport in a community of practice. This data can help EPs make informed decisions about social media engagement. Twitter is a social media platform that allows individuals to communicate through short, 280-character messages that are accessible to the public. Twitter has grown in popularity and influence among emergency physicians (EP) with over 2200 self-identified users in 2013.11As Twitter’s popularity has increased among EPs so too has its educational influence.17This study addresses this gap by analyzing the messages of influential EPs on Twitter. We sought to describe the current state of Twitter usage among EPs by exploring the tweets of influential EPs. A deep exploration of the language, frequencies, domains, and degrees of engagement of their messages can provide a contextualized understanding of the real-life Twitter experience, allowing faculty and trainees to make mindful decisions about social media participation.The purpose of this study was to describe the nature of EPs’ communications on Twitter by characterizing the language, purpose, frequencies, content, and degree of engagement of their tweets.We performed a mixed-methods analysis following a combined content analysis approach. Because text is originally qualitative, and the quantification of text alone is insufficient for successful understanding of content,19We conducted qualitative and quantitative analyses of a sample of tweets from the 61 most influential EPs on Twitter, as defined in a previous study.https://Twitter.com/search-advanced?lang=en) function to identify and download all tweets produced by the influential EPs on these random days. We stopped our analysis after 10 days of tweets when we reached theoretical sufficiency in the qualitative28To avoid variation in tweet content due to world events, national professional conferences, and seasonal variation, we analyzed tweets from random days in 2015. Specific days were identified using a random date generator function in Microsoft Excel . Once these dates were identified, we used the Twitter Advanced Search .We analyzed the content of the of tweets using a naturalistic inductive content analysis approach.After the initial code and categorical development, two team members (JY and RN) coded the remaining tweets. Any disagreements were brought to the coding team for resolution.Our experiences, backgrounds, and assumptions influence our approaches to analysis, so we chose a coding team with diverse experience with EM Twitter.To enhance the trustworthiness and credibility of our data analysis we employed memoing, reflexivity, triangulation of data among researchers, and the formation of an audit trail of the analytical process.For each individual tweet we recorded message-level data to better understand tweet engagement. We defined tweet engagement by the number of retweets, “likes” , and replies (the number of responses to a tweet prior to the author re-entering the conversation). We also logged the use of hyperlinks, embedding of media (pictures or video), and the first three hashtags (a type of metadata tag that makes it possible for others to easily find messages with a specific theme or content) per message. We also recorded the number of times each of the qualitatively-derived categories were applied to a tweet. Descriptive statistics were used to analyze this data.We analyzed 1375 unique tweets from 57 unique users, representing 93% of the influential Twitter EPs. Four (7%) influential users did not record any tweets on the sample of days analyzed. Quantitatively, a majority of tweets  had some engagement in the form of retweets, likes, or replies. The mean number of times a tweet was retweeted by another user was 2.1 (standard deviation [SD] 7.24), liked was 3.4 (SD 9.4), and replied to  was 0.8 (SD 1.4).There were 448 hashtags used, occurring in 337/1375 (25%) tweets. The most common hashtags used are displayed in The qualitative analysis identified 15 distinct descriptive categories and eight thematic categories of tweets. Descriptive categories of tweet characteristics are presented in Noteworthy thematic categories with exemplary tweets are presented in Our results provide a contextualized understanding of the real-life EM Twitter experience, enabling EPs to make mindful decisions about social media participation. While the conversation skewed to medical topics, there was a significant social component to the interactions we analyzed. Humor, networking strategies, and rapport-building messages were common, revealing a human side to the EM Twitter conversation. Although not surprising given the “social” nature of social media and physicians’ desires to connect,3437,38 The emergence of a Twitter CoOP among EM and critical care may enable relational and professional communication among colleagues who might not otherwise connect due to structural, political, or geographic barriers.39Influential EPs on Twitter also demonstrated a shared domain of interest (EM) and helped each other by sharing information and building relationships. These characteristics are consistent with traditional notions of a community of practice (CoOP).Wenger-Trayner’s metaphor of “landscapes of practice” highlights the ways in which professionals negotiate their identities among many different CoOPs.The influential EPs we studied were innovators who formed the EP Twitter community based on egalitarian principles,Future work might explore the perceived value of Twitter to the individual EPs who use the platform. While humor, sharing resources, networking, retweets, likes, and replies may appear on the surface to represent connection to the EM community, we did not explore whether Twitter users truly experienced this sense of connection. A recent study demonstrated that young adults with high social media use feel more socially isolated than their counterparts with lower social media use.Our data suggests that people are engaging in conversation and interacting by exchanging resources, creating new contacts, sharing ideas, thoughts, and reflections. While we see this broadly as a positive trend, it may be dangerous if, as has been reported, half of medical tweets from professional accounts are inaccurate.We analyzed English-language content only and findings may not generalize to the global medical community. We chose to analyze tweets from random days, allowing for the possibility that we may have missed significant and/or meaningful events in the EM community that could have changed the nature of the conversation and thus our conclusions. While we analyzed influential EPs due to the way they disproportionately impact the spread of information and directly shape social media conversations, our analysis may not reflect the lived experience of all EPs on Twitter. Further, the subjects we studied were deemed most influential from data analyzed in 2015. As EM Twitter rapidly evolves, those driving the discourse today may be significantly different from the influencers of three years ago. In particular, the representation of females on the list of influential Twitter users that we used was likely not representative of the EM social media community as a whole.Influential emergency physicians on Twitter were engaged in largely medical conversations in which most messages generated some form of interaction. They shared resources and opinions while also building social rapport in a community of practice. This data can help emergency physicians make informed decisions about social media engagement.


Amniotic fluid cytokines have been implicated in the mechanisms of preterm labor and birth. Cytokines can be packaged within or on the surface of extracellular vesicles. The main aim of this study was to test whether the protein abundance internal to and on the surface of extracellular vesicles changes in the presence of sterile intra-amniotic inflammation and proven intra-amniotic infection in women with preterm labor as compared to the women with preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection.Women who had an episode of preterm labor and underwent an amniocentesis for the diagnosis of intra-amniotic infection or intra-amniotic inflammation were classified into three groups: 1) preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection, 2) preterm labor with sterile intra-amniotic inflammation, and 3) preterm labor with intra-amniotic infection. The concentrations of 38 proteins were determined on the extracellular vesicle surface, within the vesicles, and in the soluble fraction of amniotic fluid.1) Intra-amniotic inflammation, regardless of detected microbes, was associated with an increased abundance of amniotic fluid cytokines on the extracellular vesicle surface, within vesicles, and in the soluble fraction. These changes were most prominent in women with proven intra-amniotic infection. 2) Cytokine changes on the surface of extracellular vesicles were correlated with those determined in the soluble fraction; yet the magnitude of the increase was significantly different between these compartments. 3) The performance of prediction models of early preterm delivery based on measurements on the extracellular vesicle surface was equivalent to those based on the soluble fraction.Differential packaging of amniotic fluid cytokines in extracellular vesicles during preterm labor with sterile intra-amniotic inflammation or proven intra-amniotic infection is reported herein for the first time. The current study provides insights into the biology of the intra-amniotic fluid ad may aid in the development of biomarkers for obstetrical disease. Preterm birth (spontaneous and iatrogenic) is the leading cause of perinatal morbidity and mortality –6. The kProteins present in amniotic fluid, in particular cytokines, are key regulators of parturition, and labor-associated changes in their concentrations, with or without infection at both term and preterm, have been well characterized –62. Untiin vitro ; Biological Markers of Disease in the Prediction of Preterm Delivery, Preeclampsia and Intra-Uterine Growth Restriction: A Longitudinal Study (WSU IRB#110605MP2F and NICHD/NIH# OH97-CH-N067); Microarray Expression Profiling to Identify Stereoptypic mRNA Profiles in Human Parturition (WSU IRB# 103108MP2F and NICHD/NIH # OH99-CH-N056); Cerebral Palsy: Clinical, Biochemical, Histological, and Biophysical Parameters in the Prediction of Cerebral Palsy in Patients with Preterm Labor and Premature Rupture of Membranes (NICHD/NIH# OH97-CH-N066); The Role of Feto-Maternal Inflammation as a Mechanism of Disease in the Great Obstetric Syndromes (WSU IRB# 075299M1E).Term labor was defined as the presence of regular uterine contractions with a frequency of at least 1 every 10 minutes and cervical changes occurring after 37 weeks of gestation  − [analyte content of intact vesicles].Samples and protein standards for the supernatant fluid and intact vesicles were prepared in assay buffer , and lysed samples and standards were prepared in assay buffer with TritonContinuous variables were compared among multiple groups using the Welch’s one–way analysis of variance (ANOVA)  or Krusk2 transformed to improve normality before analysis .On the surface of extracellular vesicles, median concentrations of 37 proteins were significantly increased  in preterm labor with intra-amniotic infection compared to preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection. The highest fold change (FC) was observed for MIP-3α (log2 FC = 8.7), Calgranulin A (log2 FC = 3.5), and IL-1β (log2 FC = 2.3) showed the highest increase in concentration in the presence of intra-amniotic infection.In the internal compartment of the extracellular vesicles, the median concentrations of 24 cytokines were significantly higher in preterm labor with intra-amniotic infection compared to preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection. MIP-3α , Calgranulin C (log2 FC = 2.6), and IL-1β (log2 FC = 0.9)] were significantly increased. Similarly, six cytokines  showed a significant increase in concentration in preterm labor with intra-amniotic infection compared to preterm labor with sterile intra-amniotic inflammation.2 FC >10 for all). When comparing preterm labor with sterile intra-amniotic inflammation to preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection, 20 cytokines showed significantly increased abundance, such as eotaxin-1 (log2 FC = 10.6), CXCL6 (log2 FC = 7), and Calgranulin C (log2 FC = 5.1) (2 FC = 4.9), CXCL6 (log2 FC = 4), and IFN-λ (log2 FC = 3.3), among others] also showed a significant increase in preterm labor with intra-amniotic infection compared to preterm labor with sterile intra-amniotic inflammation.In the soluble fraction of amniotic fluid, median concentrations of 37 cytokines were significantly higher in preterm labor with intra-amniotic infection compared preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection, with eotaxin-1, CXCL-6, and HMGB1 being the most increased (logC = 5.1) . Finally2-fold changes between the pairs of amniotic fluid compartments are provided in 2 fold changes on the differences observed among the amniotic fluid compartments), are summarized below:Quantile regression models were used to assess if the differences in protein abundance among preterm labor groups were significantly different among the three amniotic fluid compartments. For each pair-wise comparison among the study groups, the differences in logThe changes in protein abundance with intra-amniotic inflammation, regardless of the detection of microorganisms, were more pronounced on the extracellular vesicle surface compared to the internal compartment . Similarly, for IL-4, the change in protein abundance with intra-amniotic infection vs sterile intra-amniotic inflammation was higher within extracellular vesicles compared to that in the soluble fraction of amniotic fluid (Δ log2 FC = 1.4).The changes in protein abundance with intra-amniotic inflammation, regardless of detected microorganisms, were generally lower when assessed within the extracellular vesicles than in the soluble fraction of amniotic fluid, with a few notable exceptions , 61 delivered early preterm (< 32 weeks), and the 55 delivered late preterm (at or after 32 but before 37 weeks).For the subset of cases where amniocentesis was performed before 32 weeks of gestation, the predictive value of the compartmentalized cytokine concentrations for early preterm delivery was evaluated by building univariate and multivariate prediction models. The performance of prediction models is summarized in For 33 cytokines, the AUC estimates obtained from univariate models based on their extracellular vesicle surface concentrations were significantly higher than 0.5 . Moreover, the AUC statistic for 16 of these cytokines  was higher based on data collected on the extracellular vesicle surface than the data collected in the soluble fraction of amniotic fluid, yet the difference failed to reach statistical significance. The AUC estimates based on their extracellular vesicle internal concentrations were generally lower than the extracellular vesicle surface or amniotic fluid soluble counterparts .The performance indicators of multivariate models based on cytokine measurements in the extracellular vesicle internal, extracellular vesicle surface, and amniotic fluid soluble compartments were overall similar . The top five ranked cytokines, based on their importance in prediction, were found in these compartments: EV surface: IL-6, IL-8, MCP-1, MIP-1β, and MIP-1α; EV internal: Calgranulin C, IP-10, MIP-3α, IL-1β, and MIF; and amniotic fluid soluble: MIP-1α, MIP-1β, MIP-3α, IL-6, and IL-8. There was no significant improvement in the cross-validated point estimate of AUC of a multi-variate early preterm delivery prediction model when information from all three compartments was used [AUC = 0.86 (0.78–0.93)] as compared to individual compartments.Pathologic insults to the feto-placental unit are often clinically expressed as complications of pregnancy, such as preterm labor with intact membranes and preterm prelabor rupture of the membranes –115. A cIn addition to these canonical signaling pathways, recent studies identified the release of extracellular vesicles as a significant pathway for intercellular communication , 130. ThExtracellular vesicles regulate inflammatory and immune processes , 143–158To gain insight into the contribution of proteins packaged in extracellular vesicles, we established the profile and compartmentalization of extracellular vesicle-associated proteins in amniotic fluid obtained from women with preterm labor. We evaluated the distribution of 38 cytokines between the vesicle surface and intra-vesicle compartments. Their concentration in extracellular vesicle-free amniotic fluid was also determined for each sample. These measurements allowed us to test whether there was an advantage to measuring cytokine concentrations in the extracellular vesicle compartments, as opposed to unfractionated amniotic fluid, in predicting preterm delivery. We have specifically addressed the following research questions:We found that intra-amniotic inflammation, regardless of detected microbial invasion of the amniotic fluid cavity, was associated with an increased abundance of cytokines internal to and on the surface of extracellular vesicles. Earlier we reported on the general increase of cytokines  in unfraThe extracellular vesicle associated proteins showing the strongest increase in concentration with intra-amniotic infection were MIP-3α, Calgranulin C, CXCL6, ITAC/CXCL11, MCP1, MIP-1α, Eotaxin 1, IL-8, Gro-α/CXCL1, and IL-6 on the surface, and MIP-3α and Calgranulin A in the lumen. The up-regulation of these pro-inflammatory cytokines (IL-6), chemokines  and antimicrobial polypeptides  results from the activation of the innate immune system which includes the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors  , 162–167The top ranked proteins based on the magnitude of change in their concentrations with sterile intra-amniotic inflammation were Calgranulin C, MIP-3α, IL-16, MIP-1b, Groα/CXCL1, CXCL6, MCP-1, IL-8, CXCL13, and IL-6 on the surface of extracellular vesicles and MIP-3α, and Calgranulin C in the lumen of extracellular vesicles. The overexpression of these inflammatory mediators in the absence of microbes is initiated upon recognition of damage-associated molecular patterns  by pattern recognition receptors  , 168–171The association of intra-amniotic inflammation with or without detected microbes with preterm parturition syndrome and the role of cytokines in these processes are well documented , 180–182While cytokine changes with inflammation, regardless of detected microbial invasion of the amniotic fluid cavity, on the surface of extracellular vesicles correlated with those determined in the soluble fraction of amniotic fluid, the magnitude of the increase was significantly different between these compartments, suggesting that they include different information about the amniotic fluid milieu. Overall, although protein changes in extracellular vesicle surface and the soluble fraction in intra-amniotic inflammation, regardless of detected micro-organisms, were higher than those within the extracellular vesicles, the magnitude of changes within the extracellular vesicles was higher compared to the soluble fraction of amniotic fluid for MIP-3α.Cytokines contained within the internal compartment are not accessible to routine immunoassay quantification; therefore, this contribution has not been incorporated in contemporary models of inflammation-associated parturition. While the relative amount of internal mediators is low, their biological effect may be significant as vesicles can deliver these mediators to the very vicinity of the target cells. The roles of these extracellular vesicle -associated mediators in parturition remain to be established. Previously, we and other investigators have established that encapsulation within extracellular vesicles protects against degradation and affords alternative pathways for engagement with target cells and transport pathways .To determine the potential of amniotic fluid compartmentalized protein profiling for developing new biomarkers of preterm labor and other obstetrical complications, we have developed univariate and multi-variate prediction models for early preterm delivery . For all but one cytokine (TRAIL), the AUC estimates obtained from either the extracellular vesicle surface or the amniotic fluid soluble fraction concentrations were significantly above 0.5. The AUC statistic for univariate models based on data collected on the extracellular vesicle surface for IL-6, IL-8, MCP-1, MIP-1α, and MIP-1β was greater than 0.8. This is consistent with our previous report of the association between unfractionated amniotic fluid concentrations of these proteins and early preterm delivery in patients diagnosed with a short cervix . AlthougThe main strength of this study is its novelty component, which is the compartmentalized profiling of proteins in the amniotic fluid for three phenotypes of preterm labor. Another strength is the moderate sample size that enabled not only detection of differences among groups within a given compartment, but also detection of significant differences in the magnitude of changes between groups across the different compartments. The use of cross-validation to avoid overfitting in multi-variate models was also a strength of the analysis. Limitations are related to the variability in the gestational ages at sampling of amniotic fluid, and the lack of amniotic fluid samples collected during second or early third trimester from mothers without preterm labor.The data obtained in this study are consistent with our hypothesis that amniotic fluid proteins are differentially expressed and grouped within the extracellular vesicle and soluble amniotic fluid fraction, and compartment-specific profiles characterize clinical subgroups of preterm labor. Intra-amniotic inflammation with and without detectable microorganisms was associated with the differential packaging of extracellular vesicles cytokines. Proteins packaged within the extracellular vesicles contribute to total amniotic fluid concentrations. Although the current study points to possible improvement in biomarker prediction based on compartmentalized profiling, further de-convolution of the heterogeneity of preterm labor and delivery is warranted.S1 Fig2 transformed before plotting. Control: preterm labor without either intra-amniotic inflammation or proven intra-amniotic infection.Protein concentrations were offset by adding 1 unit and then log(TIFF)Click here for additional data file.S1 Tablewww.uniprot.org) of the genes coding for these proteins.Full names of the 38 proteins measured on the surface and within extracellular vesicles and in the soluble fraction of amniotic fluid are listed along with official symbols ((DOCX)Click here for additional data file.S2 TableEach line corresponds to one of the 33 patients with intra amniotic infection.(DOCX)Click here for additional data file.S3 TableConcentrations (pg/ml) of 38 proteins in amniotic fluid compartments  for each sample (row) are provided. ID: anonymized identifier indicator of the pregnant mother, GAatAmnio: gestational age at the time of amniocentesis (weeks); GAatDelivery: gestational age at the time of delivery (weeks); Group: preterm labor subgroup where IAI refers to intra-amniotic infection and SIAI refers to sterile intra-amniotic inflammation.(XLSX)Click here for additional data file.S4 TableThe number (and the proportion) of cases where a non-zero protein concentration was detected is presented by preterm labor group and by amniotic fluid compartment. PTL: preterm labor, EV: extracellular vesicle, AF: amniotic fluid.(DOCX)Click here for additional data file.