Chemical vapor deposition grown graphene was transferred to BN/sapphire substrates for evaluation of more than 30 samples using Raman and Hall effects measurements. A more than 2x increase in Hall mobility and 10x reduction in sheet carrier density was measured for graphene on BN/sapphire compared to sapphire substrates. Through control of the MOCVD process, BN films with roughness ranging from <0.1 nm to >1 nm were grown and used to study the effects of substrate roughness on graphene transport. Arrays of graphene field effect transistors were fabricated on 2” BN/sapphire substrates demonstrating scalability and device performance enhancement.Wafer scale (2”) BN grown by metal organic chemical vapor deposition (MOCVD) on sapphire was examined as a weakly interacting dielectric substrate for graphene, demonstrating improved transport properties over conventional sapphire and SiO Unfortunately, interactions between graphene and its environment, including the substrate, greatly degrade its transport properties. Substrate induced scattering from charged impurities, roughness, and phonons will reduce mobility3. The most successful approach to mitigate these effects has been the use of a thin van der Walls (vdW) buffer layer, like hBN5 or MoS26, preserving the mobility of the graphene to values near those of suspended material. Of the available vdW materials, hBN is one of the only insulators and due to is dielectric properties (comparable to SiO27), high surface optical phonon energy (169 eV)8 and atomically smooth surface; it has been the most successful and widely used material for preserving and protecting the intrinsic properties of 2D materials 14. The overwhelming majority of this work has used hBN flakes exfoliated from high quality bulk materials. The high purity and crystal quality of this exfoliated material has given rise to amazing properties; however, due to the small size, low yield and poor reproducibility of these exfoliated flakes this process is not scalable or suitable for device fabrication beyond the laboratory.Due to its amazing properties, graphene continues to be of great interest more than a decade after its experimental discovery18 and insulating substrates22. Taking advantage of the catalytic nature of metal substrates, like Cu and Ni, high quality material can be grown at quite low temperatures (<1000 °C). Since this process it typically surface catalyzed the controlled growth of mono-to few-layer hBN is possible23. The hBN grown on metal films can then be transferred to insulating substrates for further processing and characterization. Improved transport in graphene has been demonstrated using such transferred hBN films25 as compared to conventional substrates. Lee et al.24 demonstrated a 3x increase in graphene mobility when a few layer hBN buffer was inserted on SiO2/Si. Back gated graphene field effect transistors (FET) on hBN, grown on Fe substrates showed improved mobility and reduced intrinsic doping as compared to similar devices directly on SiO2/Si25. For many of the applications described above hBN grown on a metal will inevitably require transfer to insulating substrates adding complexity, sources of contamination, and damage from the fabrication process.The need for large area high quality hBN thin films for 2D substrates, dielectrics, and tunneling barriers has generated considerable upswing in hBN growth. High quality hBN thin films from mono to many layers thick over large areas have been produced using chemical vapor deposition (CVD) using both metallic27. The formation of large hexagonal wrinkles has also been observed in BN grown on sapphire due to the thermal expansion mismatch between BN and the substrate28. These features make BN films grown on sapphire distinct from those on metals. Jang et al. reported growth of smooth few-layer hBN on sapphire and demonstrated a 2x increase in graphene field effect mobility as compared to graphene on SiO2/Si29. Now with the availability of high quality large area CVD grown BN films on sapphire, more work is needed to explore their applications as substrates and dielectrics for 2D electronics.Alternatively, hBN films grown directly on insulating substrates do not require transfer prior to device processing. Due to the challenges of growing hBN directly on an insulating substrate, such as sapphire, it has been much less explored than its counterpart grown on metals. Growing on sapphire it is much more challenging to produce continuous ultra-thin films of mono– or bi-layer thickness, and as such, most reports demonstrate films from few to tens of nanometers thick2-BN on sapphire as a weakly interacting substrate for graphene. The basal plane of sp2 bonded BN forms the same honeycomb lattice as graphene and can form atomically thin and smooth layers. In multilayer BN, layers are known to stack hexagonal (AB stacking), rhombohedral (ABC stacking), or turbostratic (random stacking) with very small differences in inter planar spacing. For this work, CVD grown graphene was transferred to wafer scale sp2 BN/sapphire (now referred to as BN) grown by metal organic chemical vapor deposition (MOCVD) to study the effects on transport properties by Hall and FET device measurements. The effect of BN roughness was also investigated using BN films with roughness from <0.1 nm to >1 nm. Results show a more than 2x increase in graphene mobility when transferred to BN on sapphire substrate with a strong dependence on substrate induced doping related to BN roughness.In this paper, we investigate the use of few-layer sp26. Reducing the V/III ratio or increasing the pressure disrupts these self-terminating conditions resulting in thicker, rougher (>1 nm) films with a random orientation . From XPS analysis, the B/N was measured to be 1/1 to 1.05 across a range of samples. To demonstrate uniformity across each wafer Fig. 2-BN E2g mode at 5 points across 2” BN/sapphires wafers. The atomically smooth BN/sapphire is well suited for application as a substrate for 2D materials, while rougher films are useful to study the effects of substrate roughness on graphene transport.For application as a graphene substrate, BN films need to be atomically smooth, atomically flat, insulating and uniform over large areas. Growth of BN on sapphire by MOCVD at low pressure (20 Torr) and high V/III ratios (>2250) has been shown to produce films fitting these parameters with excellent thickness uniformity (1.6 + /−0.1 nm) and low roughness (<0.1 nm) over large areas Fig. . Under tion Fig. 30. Varyi2/Si as reference substrates. Raman spectroscopy of graphene films was used to compare the different dielectric substrates. Figure D/IG) taken from multiple spots on each sample was used for comparison, Fig. D/IG is 0.09 with a standard deviation (STD) of 0.05 across 6 samples. Comparable values of ID/IG of 0.1 with a STD of 0.05 were found for the samples transferred onto both the smooth and rough BN/Sapphire substrates indicating that the graphene is both uniform and undamaged during the transfer process regardless of the substrate.Monolayer graphene films grown by CVD on Cu were transferred to 20 different BN on sapphire substrates with BN roughness ranging from >1 nm to <0.1 nm to study the effects of BN roughness and morphology on graphene. Half of the BN/sapphire substrates were grown under self-terminating conditions and have measured roughness <0.1 nm. Graphene was also transferred to sapphire and SiOH) as a function of sheet carrier density (ns) for each sample. As a comparison, graphene transferred to c-plane sapphire and SiO2/Si substrates was also investigated. From this data a clear separation into groups based on the substrate can be observed, illustrating the impact the underlying substrate has on transport. Graphene films on sapphire and SiO2/Si have the lowest µH and highest ns. By switching to even a very rough (>1 nm) BN/sapphire substrate a noticeable reduction in ns and increase in µH is achieved. This trend of improving graphene transport continues as the BN roughness is reduced. A 2x increase in µH and one order of magnitude decrease in ns was achieved on the smoothest BN/sapphire substrates. The substrate is known to effect ns and µH in a number of ways causing doping, phonon scattering and/or corrugation of the graphene layer. At the surface of SiO2 and sapphire hydroxyl groups can form32, which will adsorb water and p-type dope the graphene34. Switching to the inert vdW surface of BN will eliminate this substrate doping effect assuming a near perfect surface and interface. For graphene supported by MOCVD BN/sapphire we expect a contribution to ns from surface impurities and defects, which may form hydroxyl groups similar to sapphire and SiO2. As such, it is not surprising to see a higher ns in graphene supported by rough BN. If we compare the morphologies and layer orientation  phonons3 from the substrate as well as graphene longitudinal acoustic (LA) phonon2 and short range scattering39 will all effect mobility. The mobility limited by Coulomb scattering from impurities, short range scattering from defects and roughness are all temperature independent mechanisms that will be defined here as µ0. The temperature dependent mechanisms for the graphene LA phonon (µAL) and substrate SO phonon (µSO) can be expressed as:ρsis the graphene 2D mass density, vf is the fermi velocity, vs is velocity of sound, DA is the acoustic deformation potential, E0 is the substrate phonon energy, and C is the coupling strength. Using Matthiessen’s rule, the total limited mobility is expressed as 1/µtot = 1/µ0 + 1/µLA + 1/µSO. Accordingly, we observe an inverse mobility dependence on ns 8 and sapphire (55 meV)40 shows that switching to a BN/sapphire substrate can positively impact µH at room temperature.For substrate supported graphene, scattering due to the surface impuritiess to be independent of temperature over the range of 10 to 320 K −1 shows good agreement with graphene on both smooth BN and sapphire. Only µ0, DA, C, and E0 were obtained from fitting given in Fig. A were determined to be 17.7 and 16 eV for sapphire and smooth BN substrates in good agreement with theoretical and experimental values2. The experimentally determined values for E0 are 135 meV for the smooth BN/sapphire and 60 meV sapphire. On a rough BN/sapphire substrate, graphene mobility is not well described by Eqns. . Since at higher temperatures mobility is limited by substrate induced SO phonon scattering, we fit the graphene on rough BN mobility to µtot = (1/µ0 + 1/µSO)−1 for temperatures greater than 150 K. This fit gives us an estimate of E0 (140 meV) for these BN/sapphire substrates, which is in good agreement with the smooth BN/sapphire. Using this modified version of µtot we also obtain a value of 1851 cm2/Vs for µ0, which is between those for the smooth BN and sapphire. Although, this term contains the contribution of roughness to µtot we cannot explicitly determine its contribution considering Coulomb and short range scattering are also temperature independent. If we qualitatively compare µ0 from the graphene on smooth BN and sapphire samples, which have similar roughness (<0.1 nm), we find the effects of impurity and carrier density (Coulomb and short range) to be significant suggesting a stronger contribution to µtot than roughness.Temperature dependent mobility for graphene on sapphire, rough BN, and smooth BN are shown in Fig. 2/Si FETs were also fabricated and tested. Typical Id-Vd measurements showing gate modulation in devices on smooth and rough BN/sapphire are shown in Fig. 2O3 has been shown to dope graphene and can significantly increase electron concentration, it is expected that the deposition of the Al2O3 top gate is related to this difference in majority carrier type44. The observed switch (from p to n) in majority carrier type in devices on smooth BN/graphene, but not rough BN/sapphire, is due to the lower background carrier density.Further quantifying MOCVD grown BN as a substrate for graphene devices, top-gated FETs were fabricated on a 1” graphene film transferred to a 2” BN/sapphire substrate Fig. . The mas2/Si were also studied. The Dirac point voltage is clearly different for these three dielectric substrates, Fig. 45. In our case, the bottom BN layer in the devices presented here also changes the Dirac point. Figure 46.The transfer characteristics of gFET devices on rough BN/sapphire, smooth BN/sapphire and SiOm is transconductance, defined as gm = dId/dVg, Cox is the gate oxide capacitance (Cox = κε0/tox), W is channel width, and L channel length. Using this method, we found mobility depended strongly on the channel length  graphene FET arrays were fabricated on 2” BN/sapphire and investigated showing good uniformity across the array. This collective work demonstrates significant performance improvements in graphene using scalable methods for producing BN/sapphire substrates.We have presented results from a detailed study on MOCVD BN on sapphire as a weakly interacting dielectric substrate for graphene. Wafer scale BN can be grown on sapphire with low roughness and excellent uniformity. Using Raman, Hall effect, and FET device characterization we studied the impact the dielectric substrate has on CVD transferred graphene. A greater than 10x decrease in graphene carrier density and 2x increase in mobility is achieved by using a BN/sapphire substrate as compared to SiO3 and TEB precursor gases used for N and B, respectively, are injected such that they are not heated nor do they interact with one another until they reach the surface of the substrate. The reactor was kept at a pressure of 20 Torr and the substrate was heated to 1000 °C during the growth. The ratio of NH3 to TEB was set between 2250 to 950 to achieve the desired morphology and quality of BN films.Boron Nitride thin films used in this study were grown via a metalorganic chemical vapor deposition (MOCVD) technique. The substrates used for BN growth were c-plane sapphire which allows for uniform, few layer BN to be grown. In a cold-walled vertical MOCVD reactor, the NH50. After transfer, graphene films were annealed in forming gas (5% H2: 95% Ar) for 1 hr at 300 °C to remove any additional residue from the transfer process.Graphene was grown on a copper substrate by CVD using methane as the carbon source. Films were subsequently transferred to dielectric substrates using a typical wet transfer with PMMA, similar to the process described in ref.in-situ FIB lift out technique on an FEI Dual Beam FIB/SEM and imaged with a FEI Tecnai TF-20 FEG/TEM operated at 200 kV in high-resolution (HR) TEM mode. The surface morphologies of the graphene and BN films were analyzed by AFM using a Bruker Dimension Icon in tapping mode. Raman measurements were performed using a Renishaw inVia system under a backscattering geometry with scattered radiation collected along the sapphire [0001] direction. A 4 mW 488 nm excitation source, 20 µm slits, and a 3000 l/mm grating was used for each measurement. The transport properties were characterized by Hall-effect measurements. For room temperature measurements, an Accent 5500 system was used under ambient conditions. For temperature dependent measurements from 10 to 320 K, a Lakeshore 7507 system was used. Prior to measurement the sample was loaded into the cryostat, heated to 350 K under a He atmosphere and held overnight. Measurement sweeps were taken during cooling to 10 K and heating to insure a consistent measurement. If samples were not heated to 350 K, the measurements were found to be quite different on heating and cooling.Transferred graphene and BN MOCVD layers were characterized using a suite of techniques. The BN thickness was measured by X-ray reflectance (XRR) using a PANalytical Empyrean X-ray diffractometer at a grazing incidence angle. Cross section TEM samples were prepared using the 2/Si substrates. Graphene mesas were defined using O2-plasma etch followed by source/drain metal contacts (Ti/Pt/Au) using a first lift-off process. A 20-nm thick Al2O3 was used as gate dielectric. Finally, 100-nm thick gate metal (Ti/Au) contacts were defined using a second lift-off process. Gate dimensions were chosen such that electron beam lithography process is not required for the FET fabrication process. An Agilent 4156 C precision semiconductor parametric analyzer was used for all the electrical characterization of the FETs.Top-gated graphene FETs were fabricated on transferred graphene on BN/sapphire and SiOSupplementary information


Recent years have seen a great potential of the focused ion beam (FIB) technology for the nanometer-scale patterning of a freestanding two-dimensional (2D) layer. Experimentally determined sputtering yields of the perforation process can be quantitatively explained using the binary collision theory. The main peculiarity of the interaction between the ion beams and the suspended 2D material lies in the absence of collision cascades, featured by no interaction volume. Thus, the patterning resolution is directly set by the beam diameters. Here, we demonstrate pattern resolution beyond the beam size and precise profiling of the focused ion beams. We find out that FIB exposure time of individual pixels can influence the resultant pore diameter. In return, the pore dimension as a function of the exposure dose brings out the ion beam profiles. Using this method of determining an ion-beam point spread function, we verify a Gaussian profile of focused gallium ion beams. Graphene sputtering yield is extracted from the normalization of the measured Gaussian profiles, given a total beam current. Interestingly, profiling of unbeknown helium ion beams in this way results in asymmetry of the profile. Even triangular beam shapes are observed at certain helium FIB conditions, possibly attributable to the trimer nature of the beam source. Our method of profiling ion beams with 2D-layer perforation provides more information on ion beam profiles than the conventional sharp-edge scan method does. Focused ion beams (FIBs) have been increasingly exploited in nanotechnology for more than 40 years . One of One of the most crucial aspects of ion beam profiling via the direct interaction with suspended graphene is the preparation of the ultraclean graphene membranes. First, graphene was grown on a copper foil using chemical vapor deposition (CVD) according to a previously developed method . This me+ focused ion beam. The smallest beam aperture gives a beam current value of 1.5 pA. A suspended graphene layer is then exposed in a single-pixel exposure mode. + ion dose extracted from beam current and dwell time.As a first step, freestanding graphene membranes were exposed to a Ga+ ions with 30 keV energy hit the graphene membrane, carbon atoms are sputtered at a probability of about 50% [+ beam diameter [Data for two instrument apertures corresponding to two different beam currents are presented: squares for 1.5 pA and circles for 18 pA beam current. STEM dark field (DF) images of a pore array created with 18 pA beam current are shown in the inset of bout 50% . Ga-FIBsbout 50% –6. Firstbout 50% . The maidiameter . In the + beam currents used in When focused ion beams with a certain PSF irradiate suspended graphene for a time period (dwell time), a part of the graphene layer where the exposure dose is higher than a critical dose value (defined by sputtering yield) will be removed to yield a pore of a certain radius. Therefore, a plot of inverted dwell time vs pore radius would represent the PSF of the beam. r is the pore radius, a is the height of the peak, c is the inflection point of the Gaussian curve, FWHM is the full width at half maximum, and V2D is the volume under the Gaussian bell. The “shift” parameter represents an offset of the Gaussian peak from zero and originates mainly from non-Gaussian tails of the beam. As can be seen from R2) are higher than 0.98 for both curves. The FWHM for the 1.5 pA Ga-beam is extracted to be 7.8 nm. Specifications of the instruments give 7 nm at 1.1 pA. Actually, to get this low beam current the smallest aperture has to be used. In our case the aperture was slightly worn out resulting in a higher current. This would explain the small deviation of measured FWHM from the specifications of the instrument. Interestingly, the imaging resolution with such a beam is specified to be 4 nm (determined from 35/65 edge contrast), which means that the current of less than the top 20% of the beam profile defines the imaging resolution. Another important parameter of the Gaussian curve is its bell volume, V2D. For the 1.5 pA beam current, V2D = 1.77 × 10−13 m2/s. This figure can be seen as the amount of etched graphene area per second. Knowing the atomic density of carbon in graphene, σC = 3.82 × 1019 m−2, one can calculate the amount of removed carbon atoms per unit time NC/t = V2D·σC ≈ 6.76 × 106 s−1. At the same time the number of incident Ga ions per unit time can be calculated as beam current divided by elementary charge, NGa/t = Ibeam/e = 9.4 × 106 s−1. By relating the number of sputtered carbon atoms to that of incident Ga+ ions, we can estimate sputtering yield of graphene, γC/Ga+ = NC/NGa+ ≈ 0.7. In reality, the removal of carbon atoms from the lattice leads to a lower probability of collision events during subsequent ion incidence. Thus, it leads to the decrease of sputter yield with increasing ion irradiation dose, as described in [+ beam current. In this case we measure the FWHM of the beam PSF to be 19.2 nm, which is larger than the specification of 13 nm. The graphene sputtering yield estimated in the same way as above gives γC/Ga+ ≈ 0.68. The fact that all numbers agree well with each other strengthens the model used for understanding the underlying physical phenomena. The method can be automated and used as a quick way to precisely measure Ga beam profiles in dual-beam systems.where, ribed in . The obtribed in . A simil+ ions is the low mass of He+ ions which results in much smaller sputtering yields [As the next step, we perform similar experiments with the helium focused ion beam. He FIB has several conceptual differences compared to the Ga FIB. It is based on a gas-field ion source (GFIS) that is disparate from a liquid metal ion source for gallium. GFIS features an ultra-sharp tip ending with a stable configuration of only three atoms, known as a trimer ionizing helium atoms in its vicinity . This shg yields –15. We cStrictly speaking, it is not the best way to present triangular pores in the plot where the characteristic length scale of the pores is radius. Nevertheless, we calculate the effective radius of the pore from the area of a triangular pore and assuming its round shape for completeness of the curve in V2D = 1.5 × 10−14 m2/s, the resulting graphene sputtering yield is γC/He+ = NC/NHe+ ≈ 0.09. This value agrees well with the previous report [As the next step we analyze the volume under the PSF of the 10 pA He beam. The total volume including Gaussian fit and a tail is estimated to be + beams, it is difficult to measure these pore dimensions precisely with standard STEM or HIM. Time consuming high-resolution TEM imaging would be required. The third observed limitation is the graphene membrane collapse under high current Ga+-FIBs. Actually, in the inset of + beam profiles of currents higher than a few hundred picoamperes would be difficult with the graphene-irradiation method because of the mechanical stability of the layer.As a final remark we discuss the limitations of the described method to measure the profiles of energetic focused ion beams. First, cleanliness of the graphene membranes is very important. Membranes thicker than monolayer graphene will not give an accurate relation between pore radius distribution and beam profile. Also if graphene is contaminated with organic molecules, these will decompose during the ion beam irradiation or exposure to secondary electrons and will deposit as amorphous carbon on the graphene surface competing with the sputtering process. This will result in an incorrect determination of dwell time and false beam profile curves. The second important issue is high-resolution imaging of the pores. When pore size becomes smaller than a few nanometers, like in the case of 1–3 pA HePrecise measurement of the profiles of energetic focused ion beams from their interaction with suspended graphene is demonstrated. Dependency of pore dimensions of the milled-in graphene on the exposure dose reveals information on a point spread function of the incoming ion beam. The method gives more information on the ion beam profiles than the conventional sharp-edge scan method.2/Ar gas flow (5 sccm/5 sccm) at 1000 °C for 60 min. The growth of graphene is initiated by introducing a CH4 gas flow (10 sccm) for 40 min.Graphene was grown using CVD on an Alfa Aesar 46986 Cu foil. Before the growth the foil is cleaned by Ar ion beam milling for 10 min at 250 mA and 600 V. Then, it is reduction-annealed in a H−5 Pa chamber pressure. The smallest aperture (8 μm) was used for the Ga-FIB to obtain the 1.5 pA beam current, and the third smallest aperture (25 μm) was used for the 18 pA current. He-ion beam current selected for this work was 10 pA. It was defined by the following hardware parameters: 10 micrometer aperture, 6.7 × 10−4 Pa helium pressure in the gun chamber, and a “spot” parameter between 1.8 and 2.5. SEM in the DualBeam device was used in STEM bright-field and dark-field mode (BF and DF) at 30 kV and 50 pA probe current for perforated graphene imaging. HIM at 30 kV, 0.5 pA beam current, 1 μs dwell time and 8–32 lines averaging was used for pores imaging. The pixel resolution was chosen to be better than 0.5 nm/pixel.FEI Helios NanoLab G3 UC DualBeam SEM/Ga-FIB system and Zeiss Orion Plus He-FIB equipped with a Raith Elphy MultiBeam pattern generator were used for the presented study. Both, Ga and He-FIBs were operated at 30 kV acceleration voltage and ca. 6 × 10


Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial pneumonia. The disease pathophysiology is poorly understood and the etiology remains unclear. Recent advances have generated new therapies and improved knowledge of the natural history of IPF. These gains have been brokered by advances in technology and improved insight into the role of various genes in mediating disease, but gene expression and protein levels do not always correlate. Thus, in this paper we apply a novel large scale high throughput aptamer approach to identify more than 1100 proteins in the peripheral blood of well-characterized IPF patients and normal volunteers. We use systems biology approaches to identify a unique IPF proteome signature and give insight into biological processes driving IPF. We found IPF plasma to be altered and enriched for proteins involved in defense response, wound healing and protein phosphorylation when compared to normal human plasma. Analysis also revealed a minimal protein signature that differentiated IPF patients from normal controls, which may allow for accurate diagnosis of IPF based on easily-accessible peripheral blood. This report introduces large scale unbiased protein discovery analysis to IPF and describes distinct biological processes that further inform disease biology. Idiopathic Pulmonary Fibrosis (IPF) is the most common idiopathic interstitial pneumonia and is a fatal progressive disease with a median survival of 2 to 3 years2The natural history of IPF is variable and patients can experience different and dynamic clinical courses with phenotypes ranging from accelerated disease with early mortality to slowly progressive disease67912131619© (slow off rate modified aptamers)© platform has been employed in biomarker discovery in several diseases to date26272829© measured proteins which accurately predicts disease progression in IPFStudies of the IPF proteome to date have focused on bronchoalveolar lavage fluid (BALF) and lung tissue analysis212223P < 0.0000089) (P < 4E-19) included glycogen synthase kinase-3 alpha/beta , proto-oncogene tyrosine-protein kinase Src , complement C1r subcomponent , Proprotein convertase subtilisin/kexin type 7 , cGMP-specific 3′,5′-cyclic phosphodiesterase , sphingosine kinase 1 , tyrosine-protein kinase BTK , B-cell activating factor , nascent polypeptide-associated complex subunit alpha , and GTP-binding nuclear protein Ran . Interestingly, these 10 proteins that were most significantly different between control and IPF patients were all increased in the IPF patients.The demographics and clinical characteristics of study subjects are summarized in 0000089) . The topWe next applied a secondary method to account for age differences between control and IPF cohorts. This screen identified 48 proteins which were expressed at significantly elevated or upregulated levels (≥1.5 fold) in the blood of IPF patients at screening when compared to controls . This reTo better visualize how this age-adjusted, biologically relevant protein signature differentiated the two groups, we performed hierarchical clustering on the 48 upregulated and the 116 downregulated, age-adjusted, significantly different proteins  and obstructive sleep apnea (OSA) . PrincipThe next step was to utilize our differentially expressed proteins to gain systems level insight into the disease biology of IPF. This was achieved through enrichment analysis using the online DAVID software tool. DAVID associates proteins to hierarchically clustered functional terms , and an enrichment score is calculated. The most significantly enriched processes included protein amino acid phosphorylation, VEGF signaling and intracellular signaling cascade see .We next looked at possible networks and relationships between these proteins using the ClueGo application in Cytoscape. Proteins are clustered within enriched terms  and the degree of similarity between clusters is calculated using Kappa statistics. The significantly enriched clusters included platelet activation (P = 17.0E-12), the regulation of cardiac muscle hypertrophy (P = 2.9E-6) and complement and coagulation cascades (P = 53.0E-6) . The levThe downregulated proteins were analyzed for enrichment using the DAVID online software tool. The most significantly enriched terms  included defense response, anti-apoptosis and immune response see . CytoscaWe next wanted to find a minimum set of proteins that best differentiated the healthy and IPF patients based on covariance, or relationships between proteins. This signature could potentially be used as a diagnostic tool based on non-invasive measurements made from peripheral blood. To identify the minimum multivariate protein signature that differentiated healthy and IPF patients, we used the Least Absolute Shrinkage and Selection Operator (LASSO) method as a feature selection tool, followed by Partial Least Squares Determinant Analysis (PLSDA) to assess the usefulness of the identified signature. LASSO identified an age-adjusted signature of 8 proteins that best differentiated the healthy patients from the patients with IPF. A PLSDA model of these 8 selected proteins classified the two groups perfectly, with 100% calibration accuracy and 100% cross-validation accuracy, as well as 100% sensitivity and specificity for both the healthy and the IPF groups. Latent variable 1 (LV1) was able to completely differentiate between healthy patients (negative scores on LV1) and patients with IPF  was previously employed in a project that supported a role for dysbiosis in the lung and disease progression. Alterations in the microbiome, namely an increase in Features of acute inflammation including leucocyte chemotaxis, proliferation and phagocytosis are subject to downregulation in the blood compared to normal controls in our study. Several proteins involved in regulating the response to wounding appear inhibited in the plasma of patients with IPF compared to controls. We hypothesize that this finding is indicative of the recurrent injury and loss of the alveolar epithelial barrier. The proteome findings in this study support the paradigm of recurrent injury or wounding with aberrant repair. Indeed, our findings support an intrinsic impairment of the immune response to stimuli which may, in turn, promote insufficient or even exuberant responses to improve pathogen clearance but worsen bystander damage. The response of Toll like receptors (TLRs) and other pathogen recognition receptors to pathogen associated molecular patterns (PAMPs) and danger associated molecular patterns (DAMPs) is crucial to mounting a response to infection and injury13The upregulated protein profile identified T cell co-stimulation as a process discriminating between normal and IPF patients. The role of T cell co-stimulation in regulation of lung fibrosis is controversial and complicated by the fact that measurements have been based on samples taken from different human compartments versus murine models. Studies to date have supported a role for decreased expression of inducible T cell co-stimulator (ICOS) in peripheral blood mononuclear cells (PBMCs) as a marker of disease progression and a predictor of poor survival outcomes9In vitro studies and animal models have produced robust evidence to support a central role for protein kinase activity in pulmonary fibrosis, particularly tyrosine kinase activity including platelet derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF)340424445Protein phosphorylation is a fundamental mechanism of signal transduction and is achieved by kinase activity. The high signal for phosphorylation in our upregulated proteome may represent heightened kinase activity and both these processes are enriched within the upregulated proteome. et al. demonstrated that the inhibition of both complement component C3a and C5a receptors can lead to the arrest of fibrosis and may have therapeutic potential in IPFPlatelet activation leads to the release of several profibrotic mediators and IPF patients have reported evidence of increased platelet reactivity and activation in a previous studyet al. demonstrated that the administration of recombinant LIGHT to murine models produced features of fibrotic lung disease similar to the bleomycin fibrotic phenotype, via a TSLP-dependent mechanism. Human bronchial epithelial cells challenged with LIGHT in vitro generate TSLP productionThe LASSO/PSLDA proteome signature we have identified includes novel proteins that have no previous reported associations with IPF. Armed with these target proteins however, it is interesting to speculate on their putative roles in pulmonary fibrosis. TNFSF14 (Tumor necrosis factor ligand superfamily member 14 or LIGHT) is an inflammatory molecule and a member of the TNF superfamily that our analysis also shows to be downregulated in IPF plasma compared to normal. Seemingly contradictory, the genetic deletion of LIGHT attenuates bleomycin-induced pulmonary fibrosis in animal models through the abolition of Thymic stromal lymphopoietin (TSLP) expressionThe acquisition of a distinct signature in the blood proteome of IPF patients that allows for discrimination between IPF and healthy controls is a significant proof of concept discovery. While we recognize that a blood test is not necessary to diagnose IPF patients from healthy volunteers, our work suggests that this methodology could be employed to help diagnose IPF from other forms of chronic lung disease. This will require further validation with larger numbers of patients, and exploration in other chronic lung diseases to determine whether differential signatures are producible in similar diseases. If true, the potential for change in clinical practice is considerable. The use of peripheral blood to identify disease-specific signatures may result in obviating the need for biopsy in patients who present with imaging features that are not consistent with IPF or possibly improve diagnostic confidence in patients who are not suitable for a surgical biopsy. Previous studies of plasma proteins in IPF patients identified both MMP-7 and MMP-1 as predictors of disease progression that were differentially expressed compared to normal plasmaThere are several limitations to our study. The study numbers are limited and the IPF cohort, while extensively characterized, was not subject to death over the course of 80 week follow up. This population may not be fully representative of the IPF disease spectrum and we are not able to adjust for all potential confounding variables including co-morbidities within the IPF population. The absence of a validation cohort is a weakness; however the main goal of this work was to generate hypotheses based on the proteomic data accrued. The use of slow off rate modified aptamers is novel and the aptamer results may not correlate with other protein measurement platforms. The aptamers bind to non-linear sequences with very high specificity for the selected target; this may explain some of the variance when measuring identical targets with other platforms such as ELISA31Although we did not have a validation cohort to test the accuracy of our PLSDA model, we did investigate model accuracy through cross-validation. This involved excluding a small portion of the data , building a model based on the rest of the data, and testing the accuracy of the model using the test set. By repeating this process many times and using different test sets, we were able to obtain the cross-validation accuracy by averaging the accuracy of each individual model. Thus despite the fact that there was not a validation cohort, we were still able to report a metric of model accuracy, which was calculated based on testing the model with unseen data. The final model we have reported on performed perfectly during cross-validation testing with 100% cross-validation accuracy.Our work identified biological processes that discriminate IPF from healthy controls and generates hypothesis and new targets for investigation into disease mechanisms. Our study patients were recruited to a clinical trial with the highest standards of diagnostic approach and management. The prime purpose of this work was to introduce the approach of large scale unbiased biomarker screening and the generation of subsequent mechanistic hypothesis. However, given the proposed single organ nature of IPF, the biological signal detectable in blood is dilute and may not accurately reflect ongoing change within the lung. However, peripheral blood has been employed in several biomarker studies in IPF to date68In conclusion, this work furthers the evolving evidence supporting impaired host defense as a key marker of IPF disease biology and validates some of our current understanding. We generate further hypotheses about novel potential therapeutic targets and introduce a new approach to biomarker studies in IPF. The ability to identify a minimal signature that allows clinicians and researchers alike to discriminate IPF cases from normal serves as a proof of principle that this approach may have potential in defining other forms of chronic interstitial lung disease and the further evaluation of molecular endotyping in pulmonary fibrosis.clinicaltrials.gov, clinical trials ID no. NCT01071707) . This cohort consisted of 60 patients who had samples available for analysis for at least 3 follow up time points, but this report focuses only on the baseline samples. Inclusion criteria required patients to be aged 35-80 years with a diagnosis of IPF. Exclusion criteria included a diagnosis of IPF that was > 4 years prior to screening, a diagnosis of collagen-vascular disorder, FEV1/FVC < 0.6, evidence of active infection at screening, or comorbid conditions other than IPF likely to result in death within one year. Subject follow up was for 80 weeks. Informed consent was obtained from all participating patients. The study protocol was reviewed and approved by the institutional review board of each participating center and methods were carried out in accordance with the relevant guidelines and regulations. Participating centers included: University of California Los Angeles. Los Angeles, CA, United States–University of California, San Francisco. San Francisco, CA, United States–National Jewish Medical and Research Center, Denver, CO, United States–University of Chicago, Chicago, IL, United States–University of Michigan Ann Arbor, MI, United States–Cleveland Clinic Foundation, Cleveland, OH, United States–Temple University, Philadelphia, PA, United States–Brown University, Providence, RI, United States–Vanderbilt University, Nashville, TN, United States. Patients were enrolled from March 2010 to March 2011. Blood samples and demographic data were also acquired from healthy human controls (n = 21). Demographics are displayed separately for IPF patients and healthy normal participants, with mean and standard deviation for the continuous predictor age and the number and percentage enrolled for the categorical variable gender. Statistical significance of differences between the two groups of people for age and gender were assessed via Student’s t test and Pearson’s Chi-squared test, respectively . Plasma samples were diluted at 3 different concentrations for analysis on the aptamer array at the optimal concentrations for each SOMAmer© analytes may be found online .The SOMAscan® proteomic assay has been described extensively in previous publicationsProteomic data is reported quantitatively as RFU’s for 1129 analytes in 60 IPF patients and 21 healthy controls. For a graphic summary of our investigative approach see The initial approach first identified 203 proteins that differentiated IPF from controls. Relative fold change in blood protein levels were calculated by dividing the average intensity in IPF samples by the average intensity in the healthy samples. Statistical analysis between the healthy and IPF patients was performed by a standard two-tailed and two-sample t-test. Graphical representation of the proteomic data was created using GraphPad Prism software . Significantly different proteins were those that passed a set false discovery rate threshold of 1%. Hierarchical clustering of significantly different proteins was generated by unsupervised average linkage hierarchical clustering using Pearson’s correlation coefficient as the distance metricUpon comparison of epidemiological factors between the two groups, we found age to be slightly increased in the normal group. To account for this and identify age-adjusted proteomic differences, we performed linear regression with all biomarkers and age as predictors based on comparison between the IPF and normal cohort, and assessed mean analyte differences between IPF patients and controls adjusted for age. To account for multiple comparisons, we considered Benjamini-Hochberg false discovery rate methods58http://david.abcc.ncifcrf.gov/)62uniprot_accession as identifier and gene list as list type, medium stringency and Bonferroni correction was applied. Enrichment chart analysis was performed using Gene Ontology (GO) biological processes (BP FAT), GO molecular function (MF FAT) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The top functional annotation clusters with significant enrichment scores were identified.To identify significantly enriched biological process that differentiate IPF from control, those proteins that passed initial screening steps (a Bonferroni correction and linear regression modelling for age) were catalogued into “upregulated” and “downregulated” profiles. In brief, proteins that were meaningfully “upregulated” or “downregulated” were deemed to have potentially significant biological roles in IPF patients compared to the control cohort. A fold increase over control mean of 1.5 and a fold decrease below control mean of 0.75 were used as thresholds for “upregulated” and “downregulated” proteins, respectively. These criteria selected out 48 upregulated proteins and 116 downregulated proteins when comparing IPF patients to controls  and S3. 60K-fold cross-validation was used to generate the model that had the lowest possible mean squared error for prediction. Associated features for this model were chosen as the minimum set of biomarkers. In order to allow for age-adjustment in the LASSO model, age was forced into the model as a parameter and assigned zero penalty. PLSDA assessed the usefulness of the LASSO-identified protein signature for differentiating healthy and IPF patients. Data were normalized with mean centering and variance scaling, and cross-validation was performed by iteratively excluding random subsets in groups of 9-10 data points during model calibration. Excluded data samples would then be used to test model predictions. Hierarchical clustering of LASSO-identified proteins was generated by unsupervised average linkage hierarchical clustering using Pearson’s correlation coefficient as the distance metric.The Least Absolute Shrinkage and Selection Operator (LASSO) methodTo investigate whether or not the comorbidities present in some IPF patients affected the feature selection by LASSO or the clustering in PLSDA, we performed a principal component analysis (PCA) on all of the measured blood proteins in the healthy and IPF patients. PCA was chosen as the method of analysis due to the lack of knowledge of the comorbidities seen within the healthy cohort. Gastroesophageal reflux disease (GERD) and obstructive sleep apnea (OSA) were examined based on their prevalence in the IPF patients (34 patients with GERD and 12 patients with OSA).How to cite this article: O’Dwyer, D. N. et al. The peripheral blood proteome signature of idiopathic pulmonary fibrosis is distinct from normal and is associated with novel immunological processes. Sci. Rep.7, 46560; doi: 10.1038/srep46560 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


FOXC2 and GJC2 have reduced venous valve number and leaflet length. Experiments in mice by Lyons et al. show that Foxc2-Calcineurin-Nfatc1, and Gja4, Gjc2, Gja1 regulate valve-forming cell organization. Foxc2, Calcineurin-Nfatc1, and blood flow regulate leaflet growth/maturation.Patients with mutations in  FOXC2 and GJC2 mutations are associated with reduced VV number and length. In mice, early VV formation is marked by elongation and reorientation (“organization”) of Prox1hi endothelial cells by postnatal day 0. The expression of the transcription factors Foxc2 and Nfatc1 and the gap junction proteins Gjc2, Gja1, and Gja4 were temporospatially regulated during this process. Foxc2 and Nfatc1 were coexpressed at P0, and combined Foxc2 deletion with calcineurin-Nfat inhibition disrupted early Prox1hi endothelial organization, suggesting cooperative Foxc2–Nfatc1 patterning of these events. Genetic deletion of Gjc2, Gja4, or Gja1 also disrupted early VV Prox1hi endothelial organization at postnatal day 0, and this likely underlies the VV defects seen in patients with GJC2 mutations. Knockout of Gja4 or Gjc2 resulted in reduced proliferation of Prox1hi valve-forming cells. At later stages of blood flow, Foxc2 and calcineurin-Nfat signaling are each required for growth of the valve leaflets, whereas Foxc2 is not required for VV maintenance.Venous valves (VVs) prevent venous hypertension and ulceration. We report that  Venous valves (VVs) are widely distributed throughout veins and venules and facilitate unidirectional blood flow back to the heart, which acts to reduce the peripheral venous blood pressure . VV failItga9, Efnb2, Fn-EIIIA) regulating lymphangiogenesis also control VV formation and maintenance (FOXC2) CX47 (GJC2), and CX43 (GJA1) cause primary lymphedema in man (−/−Gjc2 and −/−Gja4 (Cx37) mice have VV defects during VV maturation, but the timing of onset of those abnormalities during VV formation and the developmental processes underlying absent VVs have not, to our knowledge, been studied  has not been elucidated . We haventenance . Severalntenance . Mutatioa in man . Gjc2−/− studied , 2016. E studied . Similar studied .GJC2 or FOXC2 and examined the earliest underlying mechanisms of VV failure caused by loss of those genes in mice. We identified reduced numbers of VVs and shorter VV leaflets in patients with lymphedema caused by mutations in FOXC2 or GJC2. In mice, we characterized the induction and organization of valve-forming ECs (VFCs), which occur at an earlier point than previously identified, and show that they occur within highly spatially and temporally regulated domains of Prox1, Foxc2, Nfatc1, Cx37, Cx43, and Cx47 expression  resulted in the failure of early VFC organization. Loss of connexin-encoding genes Gja1, Gjc2, or Gja4 similarly resulted in a failure of VFC organization, and for Gjc2 and Gja4, that resulted in reduced VFC proliferation. Both Nfat and Foxc2 signaling and blood flow were required for VV maturation to postnatal day 6 (P6). Unlike its role in LVs, we show that Foxc2 is not required in VV ECs for valve maintenance  and for leaflet length in repeated measurement by different operators . This allowed quantification of the number of VVs and VV leaflet length in the upper and lower limbs (Tables S1 and S2). To limit scan duration, we scanned only the short saphenous, popliteal, basilic, and brachial veins. Consistent with postmortem studies, different numbers of VVs (Tables S2 and S3) were detected in control veins  and, as expected, with different leaflet lengths  and GJC2 . VV defects were present in the veins of both upper and lower limbs in patients for both FOXC2 and GJC2 (not depicted). There also appeared to be fewer VVs in the single patient identified with lymphedema caused by a mutation in GJA1 (CX43). Consistent with these findings, and in agreement with their expression pattern in mouse VVs, FOXC2, and CX43 were localized on adult human VVs, suggesting that human VV phenotypes may result from tissue-autonomous effects of the patients’ mutations , which damages VV structures and precluded analysis of the anterior vein wall and complete staging of VV development  because of the critical role of proximal deep VV in human disease and because a VV most consistently develops at this position in the mouse, allowing reliable identification of the earliest stages in its development . We had elopment . We therhi” cells . Organization of VFCs is more prominent on the anterior wall of the FV, before full circularization  in combination with floxed target alleles (lxFoxc2/+) or homozygous (lx/lx)Foxc2 deletion produced a significant phenotype at P0  in venous endothelium from E15, or administration of cyclosporin A  from E17 resulted in the expected cytoplasmic localization of Nfatc1  but had no effect on VFC organization at P0 . At P0 Cx37 was localized to VFCs, particularly in the upper and lower region of the vessel (Gja4 (Cx37) homozygous constitutive KO mice, initiation of VV formation at the usual location and the typical boundary between higher distal Prox1 immunostaining and higher proximal Foxc2 immunostaining was preserved. VFCs were identified distributed across the full width of the vessel but appeared highly disorganized, failing to reach stage 1, whereas all WT and heterozygous KO littermate VVs reached stage 1, as expected  resulted in the failure of organization of the VFCs downstream of that region, and VVs failed to reach stage 1, whereas WT littermates developed normally  reporter mice, with signal amplification using antibodies raised against GFP. Although no GFP signal was detected in the FVs of WT mice, expression was detected in cells dispersed throughout the FV in GFP/+Gjc2 mice . Compared with WT littermates, Prox1hi VFCs showed a pattern of disorganization similar to loss of Gja4 and Gja1 at P0, but we could find very few VFCs expressing detectable Cx47  can be used as a surrogate marker for cellular morphology in P0 VFCs  resulted in a reduced proportion of Prox1hi nuclei in the center of the vessel  at P0  at P0 , or delec2 alone  were eacen at P6 , demonstPpp3r1, Foxc2, and to some extent, Itga9 or Efnb2 , analysis of −/−Gja4 VVs at P6 revealed complete absence of both leaflet structure and Prox1hi or Foxc2hi cells (−/−Gja4 (Cx37) mice is subsequently lost, and Cx37 is required to maintain the free-edge cell phenotype. This was associated with failure to form the gap in smooth muscle actin (SMA)–expressing cells normally seen around the valve . These results are consistent with previously reported findings in LVs and with the absence of VVs at P4 in −/−Gja4 mice .At P2, Cx43 was barely detectable in VV leaflets , but at P6, it was clearly localized around Nfatc1-expressing ECs . ConditiGFP/+Gjc2 VVs at P2 , but by P6, Cx47-expressing cells were clearly detected within the valve (GFP/GFPGjc2 mice (lacking Gjc2 expression) had no valve cells by P2, indicating loss of the Prox1hi phenotype seen at P0. These results suggest that the critical requirement for Cx47 is restricted to events around P0 , are up-regulated by fluid shear stress, we hypothesized that blood flow may be required for normal VV leaflet growth , but interestingly, VV length at 6 wk (lx/+Foxc2 or lx/lxFoxc2) or 28 wk (lx/+Foxc2) was similar to littermate controls  and one in the intracellular loop (GJA1 (Cx43) results in a substitution (K206R) in the highly conserved SRPTEK motif of the second extracellular loop and may exert dominant negative effects on connexin docking, gap-junction function, and levels of WT protein  in 4-wk-old mice, suggesting that Foxc2 depletion in VVs was efficient. In our experiments, we either deleted Ppp3r1 (CnB1) or administered CsA. Although we did not observe specific staining in developing VVs using antibodies raised against Nfatc2, Nfatc3, or Nfatc4 (unpublished data), we cannot fully exclude possible roles for these proteins.In lymphatic endothelium, Foxc2 and Nfatc1 are coexpressed, physically interact, and signal cooperatively to pattern vessel maturation . We examhi VFCs, Cx43 and Cx47 were predominantly expressed by cells upstream of the VFCs, rather than by VFCs themselves. Despite that, loss of Gja1, Gjc2, and Gja4 all resulted in failure of organization of the early Prox1hi VFCs, characterized by loss of Prox1hi elongation and reorientation. Cx37 loss resulted in a lower proportion of VFCs in the central portion of the vein, but that was not seen for loss of Cx43 or Cx47. Loss of Cx37 or Cx47 resulted in reduced VFC proliferation. It remains unknown whether this is a direct effect of the loss of gap junctions or because of the failure of VFCs to organize normally, which could lead to reduced cell–cell signaling and loss of reinforcement of VFC identity. The loss of proliferation seen with Gja4 KO is surprising because in other settings loss of Cx37 was associated with increased proliferation (hi cell organization (and resulting breakdown of inter-VFC communication) that results in the subsequent failure of further valve development in these mice.Although variation in connexin expression in veins at P0 has recently been reported, the expression was not related to positioning at the sites of VV formation, which was not described until P4, when VV leaflets are already maturing . Here, wferation . Althoughi) VFCs at P0 shows the presence of gap junctions. It is possible that groups of ECs could develop discrete regions of gap-junction intercellular communication, for example, upstream of the initial Prox1hi ring  or between free-edge cells themselves (Cx37), allowing for patterning of development by restricted regional endothelial signal propagation (hi cells. The population(s) of cells contributing to VV have not been defined and could include contributions from that upstream region, for example, by becoming leaflet ECs or by communicating with valve cells to regulate their behavior. Interpretation of connexin phenotypes is further complicated by the formation of heteromeric and/or heterotypic channels  or CnB1 (when a strikingly complete VFC ring remained). This restricts the requirement for Nfatc1 to extension of the VV leaflets and not to maintenance of VFC identity. Our results suggest that Cx37 and Cx47 are required for the early stability of the VFC ring phenotype, as has previously been suggested for Cx37 in LVs  could regulate VVs and compensate for loss of FOXC2 .The following veins underwent ultrasonographic evaluation: the brachial , basilic , popliteal (adductor hiatus to trifurcation), and short saphenous . Veins were visualized along their entire length, switching between longitudinal and transverse views to detect VVs, using a Philips Healthcare IU22 ultrasound machine with L17-5MHz/L9-3MHz probes. Participants with any history of deep vein thrombosis were excluded as were ablated or operated veins. Images and cine loops were recorded of each VV in B mode and with color Doppler. VV maximum leaflet measurements were obtained offline . For each vein, the number of VVs and VV length was normalized to the mean value in the respective control veins.FOXC2 mutations was performed by the South West Thames Regional Genetics Service at St George’s Hospital, University of London. Screening for GJC2 and GJA1 mutations was performed as previously described  was injected i.p. with either 5 mg at E15 for analysis at P0, or 50 µg at P0 for analysis at P6  was administered i.p. twice per day at P1–P6, and VVs were analyzed at P6. For combined deletion of Foxc2 and calcineurin inhibition, deletion was performed from E15, and CsA at 50 µg/g mouse weight i.p. twice per day from E17 to E19. For induction of deletion at 4 wk, mice received 1 mg Tam i.p. once per day for 2 d. For flow alteration at P0, pups were anaesthetized (Univentor 4000) with isoflurane, and the femoral artery and vein (which are inseparable at P0) were ligated (10/0 vicryl), and the vein was divided before closure (10/0 vicryl); postoperative care was provided with the mother.In brief, BALB/c, MF1, FVB, C57BL/6, and CD1 WT mice were obtained from Charles River UK. WT analyses were performed in BALB/c mice, unless indicated. 1CreERT2 , Foxc2lx before fixation overnight in glutaraldehyde  and postfixation in osmium tetroxide  for 1.5 h. For visualization of mouse gap junctions, samples were immersed in 1% osmium tetroxide: 1.125% potassium ferrocyanide for 1 h, followed by en block staining with 1% aqueous uranyl acetate. All samples were dehydrated through graded ethanols, equilibrated with propylene oxide, infiltrated with epoxy resin (TAAB Laboratories Equipment), and polymerized at 70°C for 24 h. Semithin sections (0.5 µm) were cut and stained with 1% Toluidine Blue. Ultrathin sections  were mounted and contrasted using uranyl acetate/lead citrate for examination .For scanning electron microscopy (SEM), pups were culled and perfused via the aorta with hPBS before opening the femoral and iliac veins and overnight fixation at 4°C in glutaraldehyde  and postfixation in 1% (wt/vol) osmium tetroxide for 40 min at room temperature. After washing, samples were dehydrated through graded ethanols before critical point drying  and mounting with conductive carbon cement (TAAB Laboratories Equipment) on aluminum pins (TAAB Laboratories Equipment) and gold sputter coating  for examination . VV leaflet length and position were measured in National Institutes of Health (NIH) ImageJ software.4Cl, blocking in 2% BSA, and incubation with primary antibody. For localization of FOXC2, after fixation in −20°C acetone and quenching of endogenous peroxidase using 3% H2O2, blocking , and incubation with primary antibody, signal was amplified using polymer-HRP , according to the manufacturer’s instructions. Localization of integrinα9 was performed on formalin-fixed, paraffin-embedded sections, using tyramide amplification (PerkinElmer), according to the manufacturer’s instructions.For localization of connexins in human VVs (obtained from patients undergoing coronary artery or lower limb bypass grafting), 10-µm frozen sections were thawed and fixed in −20°C MeOH before permeabilization in 0.2% Triton X-100, charge neutralization in 0.5 M NHMice were culled and perfusion fixed via the aorta and femoral vein with hPBS, followed by fixation with 4% formaldehyde, and then further fixed for 24 h. The external iliac and femoral veins were excised with surrounding muscles and processed for paraffin wax embedding and hematoxylin-eosin stains. 5-µm sections were photographed using a MicroPublisher 3.3 real-time viewing camera mounted on a Leitz DMRB microscope with PL Fluotar ×10, ×20, and ×40 lenses (Leica). Maximum leaflet length of each valve was measured in NIH ImageJ.hi nuclear elongation (length/width ratio) and reorientation (relative to the long axis of the vessel) were quantified in z-projections in ImageJ, as previously described  and presented as a proportion of total Prox1hi VFCs. A reduced z-projection  is shown  between sequential incubation with antibodies raised against Nfatc1 followed by Foxc2. Samples were then further dissected and mounted in Prolong Gold (Thermo Fisher Scientific). The consistent site of VV formation in unopened mouse FV was visualized by confocal microscopy  to produce Z projections  of median filtered  stacks. VV Prox1escribed . Reorienis shown .Antibodies were raised in rabbits to Cx43 , sheep to FOXC2 , Foxc2 , and goat to Integrinα9 .Antibodies were raised in rabbit to Cx43 , Cx37 , Prox1 , cleaved caspase 3 , Ki-67 , sheep to Foxc2 , goat to Nfatc1 , Prox1 , and GFP  rat to PECAM1 (BD clone MEC 13.3) and mouse to α-SMA (clone 1A4 conjugated to Cy3 [Sigma-Aldrich] or FITC [Abcam]).Secondary antibodies or streptavidin were conjugated to Dylight-405/488/550/649  and streptavidin-HRP/alkaline phosphatase (Dako). IgG controls were nonimmune sheep/rabbit/goat IgG (R&D Systems).2/Fisher’s exact test). For quantification of connexin-deletion phenotypes  and Fisher’s exact test. All comparisons of human VV disease phenotypes were performed using ANOVA with Bonferroni correction. In mice, for VV developmental-stage 0–4 quantification, data represent the proportion of analyzed valves reaching each developmental stage. P-values represent differences in the proportion of valves at each stage versus WT littermates . VV area was compared using an independent-samples t test. Abnormal VV development after CsA administration to P6 was compared by χ2 test. For VV maintenance, an independent-sample t test was used for two groups, and ANOVA was used for three groups. P < 0.05 was considered significant. All analyses were performed using IBM SPSS software (version 22).For analysis of leted VV , leafletFurther details including patient characteristics, VV variability in mouse strains, and individual channels for multi-channel images are provided in online supplemental materials. Fig. S1 shows the variability in valves in different strains of mice. Figs. S2 and S3 show the regulation of initial VV organization . Fig. S4 shows the quantification of connexin phenotypes. Fig. S5 shows the maturation of leaflets and commissures. Table S1 shows the characteristics of study participants, and Table S2 shows the number of valves per vein in the control group, whereas Table S3 provides leaflet lengths per vein in the control group.


P = 0.0087). This pathology was not significantly associated with pertinent clinical, genetic (c9ALS) or nervous system pathologic data, suggesting it is not limited to any particular subgroup of ALS patients. Among 25 non-ALS muscle samples, pTDP-43 inclusions were seen only in the autophagy-related disorder inclusion body myositis (IBM) (n = 4), where they were more diffuse than in positive ALS samples (P = 0.007). As in IBM samples, pTDP-43 aggregates in ALS were p62/ sequestosome-1-positive, potentially indicating induction of autophagy. Phospho-TDP-43-positive ALS and IBM samples also showed significant up-regulation of TARDBP and SQSTM1 expression. These findings implicate axial skeletal muscle as an additional site of pTDP-43 pathology in some ALS patients, including sporadic and familial cases, which is deserving of further investigation.Muscle atrophy with weakness is a core feature of amyotrophic lateral sclerosis (ALS) that has long been attributed to motor neuron loss alone. However, several studies in ALS patients, and more so in animal models, have challenged this assumption with the latter providing direct evidence that muscle can play an active role in the disease. Here, we examined the possible role of cell autonomous pathology in 148 skeletal muscle samples from 57 ALS patients, identifying phosphorylated TAR DNA-binding protein (pTDP-43) inclusions in the muscle fibers of 19 patients (33.3%) and 24 tissue samples (16.2% of specimens). A muscle group-specific difference was identified with pTDP-43 pathology being significantly more common in axial  than appendicular muscles (The online version of this article (10.1186/s40478-018-0528-y) contains supplementary material, which is available to authorized users. ALS is a progressive disorder characterized by motor neuron injury, muscle weakness, bulbar symptoms, such as dysarthria and dysphagia , and froPathologic studies of ALS nervous tissue have emphasized cell autonomous and non-cell autonomous pathologies in upper and lower motor neuron groups, and more recently, in non-motor brain regions , 38. Themus), but not in nervous tissue, develop neurologic and pathologic ALS [Studies in ALS animal models have provided direct evidence that skeletal muscle can play a more active role in neurodegeneration. A variety of cell autonomous pathologies have been identified in the myofibers of transgenic (tg) mice expressing mutant Cu/Zn superoxide dismutase (mSOD1) . These iogic ALS , with thogic ALS  and motoogic ALS .To further examine whether cell autonomous pathology is present in the muscle of human ALS patients, we studied 148 muscle specimens from 57 patients to identify the prevalence and muscle group distribution of pTDP-43, a well-characterized marker of ALS pathology in motor neurons. To extend the work of prior studies, we included samples not only of appendicular muscle , but also diaphragm and paraspinous muscle groups. We also studied pTDP-43-positive samples to determine whether the autophagy-related protein p62/ Sequestosome-1 was present in myofiber inclusions. To determine the specificity of these findings, we compared the ALS samples to an additional 25 non-ALS muscle samples with neurogenic atrophy, including four samples of clinical and pathologic inclusion body myositis (IBM). Finally, we examined whether pTDP-43 pathology in muscle was associated with salient disease characteristics, including age, site of onset, disease duration, familial or c9ALS status, and the burden of nervous system TDP-43 pathology.The 57 ALS patients with tissue examined in this study all underwent autopsy at our institution. The sample was representative of clinically diagnosed ALS patients seen at our institution with (1) pathologic confirmation and (2) skeletal muscle samples obtained at autopsy from one or multiple muscle groups. All pathologically confirmed ALS cases with skeletal muscle available for study were included. The majority of patients were evaluated in the clinic of two study authors . The study was conducted with the approval of the Institutional Review Board at Houston Methodist Hospital (IRB-(3 N)-0114–0013).Clinical variables recorded included (a) disease duration, defined as the time between first symptoms and autopsy date, (b) age at death, (c) site of disease onset , and (d) whether the patient had familial (fALS) or sporadic ALS (sALS). As previously described , c9ALS sn = 25) included seventeen samples with neurogenic atrophy demonstrated on muscle biopsy. These non-ALS patients comprised four cases of biopsy-proven inclusion body myositis (IBM), one case with neurogenic atrophy in a patient with myasthenic symptoms, one case of denervation atrophy in a patient with nerve plexus injury, one case of steroid myopathy, and 10 biopsies with a range of mild neurogenic changes . Eleven of the non-ALS biopsies were from quadriceps and the remainder were obtained from biceps (2), gastrocnemius (2), or an unspecified site (2). An additional eight samples were included that contained fairly abundant paraspinous muscle among the various tissues obtained at laminectomy and submitted for routine pathologic examination. Two such specimens were from cervical spine with the remainder from lumbar spine and diagnoses included synovial cyst (2), radiculopathy (1), spinal stenosis (4), and spondylolisthesis (1).Non-ALS patients (n = 44), deltoid (n = 34), diaphragm (n = 29), quadriceps (n = 26), skeletal muscle, NOS (n = 13), and one sample each of gastrocnemius and abdominal wall muscle. Immunostaining was performed on formalin-fixed, paraffin embedded (FFPE) tissue samples, sectioned at 4 μm, mounted on charged slides and dried for at least 2 h at 50–60 °C. Deparaffinization and rehydration were carried out using a series of xylenes, graded alcohols, and reagent grade water . Heat-based antigen retrieval was performed using a 1× antigen retrieval solution at pH 9  and was carried out for 1 h . Subsequent washing steps were carried out using a commercial Tris-buffered saline solution (1×) containing Tween 20, pH 7.6 (Agilent Technologies) with a 3% hydrogen peroxide solution  used to block endogenous peroxidase. Primary antibody was applied for a minimum of 1 h following a one-hour blocking step at room temperature with 2.5% horse serum . Slides were thoroughly washed and a species appropriate ImmPress™ HRP IgG detection kit (Vector Laboratories) was applied for 1 h at room temperature. Following additional washing steps, target antigen was visualized using DAB chromogen in substrate buffer (Agilent Technologies). Hematoxylin counterstain was applied and slides were taken to xylene and mounted with Permount™ (ThermoFisher Scientific). Negative and positive controls were used with all staining reactions. Positive controls comprised ALS spinal cord or medulla and performed appropriately.Phospho(409/410)-TDP43 antibody  was applied to all ALS samples, including paraspinous muscle . For pTDP-43 extent, the number of LPFs with pTDP-43 pathology per slide was expressed as a percentage of all LPFs with myofibers in the sample .Myofibers, adipose tissue, and neurovascular tissue were assessed for pTDP-43-reactive inclusions. For myofibers, only cases with unequivocal staining in the subsarcolemmal region or within sarcoplasm were recorded as positive (no intranuclear inclusions were identified). Semi-quantitation of pTDP43-positive samples was also performed. For pTDP-43 Using the procedures described above, skeletal muscle samples that were positive for pTDP-43 were screened for inclusions of p62  and/or FUS/TLS . Immunohistochemical procedures were identical to those described above. Negative and positive controls were used and performed appropriately. For p62, sections of spinal cord or medulla from ALS patients served as positive controls. For FUS, positive controls were sections of FTLD-FUS brain tissue.In a subset of ALS cases with pTDP-43 inclusions, double labeling immunofluorescence procedures were performed to assess co-expression of pTDP-43 (as above) with fast myosin , slow myosin , or p62 (as above). Samples were incubated overnight with a mixture of primary antibodies at 4 °C following deparaffinization and rehydration steps, antigen retrieval procedures (10 mM citrate buffer), and a blocking step using 2.5% horse serum. After several washes with phosphate buffered saline, secondary antibodies (1:200) were applied for 1 h, including the secondary antibodies Alexa Fluor® 555 anti-rabbit IgG , Alexa Fluor® 555 anti-mouse IgG , Alexa Fluor® 488 anti-mouse IgG , and Alexa Fluor® 488 anti-rabbit IgG . Following further PBS washes, sections were mounted using Vectashield Antifade mounting media with DAPI .Additional staining for N-terminal TDP-43 antibody , which stains both physiologic and pathologic TDP-43, was performed in three ALS samples with pTDP-43 inclusions see , two ALSAll immunofluorescence studies were reviewed within 24 h of staining and images were captured in cellSens software 1.13  on an Olympus BX-43 Microscope using a DP71 camera, a EGFP FITC/Cy2 filter cube  and a CY3/TRITC filter cube . Slides were examined separately under DAPI, TRITC, and FITC filters, to facilitate examination of antibody/ fluorophore-specific signal versus structures with autofluorescence . Images were captured in cellSens software, including merged images combining all three channels.oC. Semi-thick and ultrathin sections were collected using a LEICA EM UC7 . Stained thin sections were imaged with JEOL 1400 transmission electron microscope  and AMT Imaging Software .A protocol at our institution available for the ultrastructural study of specimens from FFPE blocks not fixed with glutaraldehyde was used to study four ALS muscle samples with comparatively extensive pTDP-43 inclusion pathology see . A regioSQSTM1 (p62/ sequestosome-1) and TARDBP gene expression, RNA was isolated from four successive 10-μm sections of paraffin-embedded muscle tissue using an RNeasy® FFPE Kit  according to the manufacturer’s protocol. RNA was isolated from five ALS samples (p62 and pTDP-43-positive), three IBM samples (p62 and pTDP-43-positive), and three samples of non-ALS, non-IBM neurogenic atrophy (p62 and pTDP-43-negative). Quantification of RNA was performed using a Nanodrop ND 100 spectrophotometer (Thermo Fisher Scientific).To examine SQSTM1  and TARDBP . Housekeeping genes utilized for normalization of transcript levels included GAPDH  and RPS18 . Each real-time PCR experiment included eleven ALS, IBM, and atrophy muscle RNA samples plus a no template control . This experiment was duplicated in the other half of the 96-well plate and then repeated, again in duplicate, using a new 96-well plate. A one-step real-time PCR protocol was performed on a CFX96 Touch , including a melt curve step to exclude non-specific amplification. Experiments were analyzed in CFX Maestro™ software for Mac . TARDBP and SQSTM1 expression were normalized to both reference genes  and relative normalized gene expression was compared between patient groups. Significance testing was performed using an unpaired t-test.Real-time PCR primer assays were performed using SYBR® Green dye-based chemistry  with commercially available and experimentally validated PCR primer pairs for P-value and odds ratio (OR).Fisher exact tests were used to determine the strength of association between pTDP-43 inclusion pathology in muscle fibers and individual muscle groups , as well as axial  versus appendicular  musculature. Fisher exact tests were also used to examine the association between pTDP-43 inclusion pathology and c9ALS status, fALS status, and onset (limb versus bulbar). The strength of these associations was recorded by the P ≤ 0.01.Wilcoxon rank sum tests (Mann-Whitney U) were applied to examine the equality of medians in pTDP-43-positive and negative ALS patients with respect to age at death, disease duration, and percentage of central nervous system regions-of-interest (ROIs) positive for TDP-43 pathology . As prevn = 45, 79%), or family history was unknown (n = 2). c9ALS was present in 13 patients (22.8%), accounting for 90% of the clinically designated fALS patients in this study and 4 sALS patients (8.9% of sALS cases). Limb onset was seen in 38 patients (66.7%), bulbar onset in 12 (21.1%), and onset was other/ multifocal in six (10.5%). Onset site was not available in one patient. Median disease duration was 2.99 years .ALS patient characteristics are listed in Table . Intranuclear inclusions were not identified.pTDP-43 inclusion pathology was present in 24 of 148 ALS muscle specimens (16.2% of blocks) from 19 of 57 patients (33.3% of patients). pTDP-43 was identified in myofibers as cytoplasmic or subsarcolemmal aggregates, ranging from blocky, immunoreactive structures to dot- and dash-like and filamentous inclusions. Representative images of p62 and pTDP-43 pathology are shown in Fig. Quantitatively, a range of inclusion density and spatial extent was seen across pTDP-43-positive ALS samples. The median number of pTDP-43 inclusions per 100× field was 2 . The majority of positive samples had multiple LPFs of pTDP-43 pathology, with a median of 5 LPFs per case having pTDP-43 inclusions . A median of 10% of all microscopic LPFs that included skeletal muscle had pTDP-43 pathology (range of 3%–60%), reflecting that the inclusion pathology was patchy and not typically diffuse.Co-localization of pTDP-43 inclusion pathology was examined with respect to fast- and slow-myosin heavy chain expression in 5 ALS cases and 1 IBM sample. In ALS cases, a median of 2 pTDP-43-immunoreactive inclusions was seen in the positive high-power field studied (range of 1–14). Phospho-TDP-43 inclusions in both slow- and fast-myosin positive fibers were seen in two cases . The remaining ALS cases had pTDP-43 positive inclusions only in slow fibers (2 cases) or fast fibers (1 case). The single IBM sample tested had 83.3% of pTDP-43-positive inclusions in myofibers expressing fast myosin.All but one pTDP-43-positive ALS sample (23 of 24 samples) had pathologic inclusions of p62 in myofibers in the same distribution Fig. . As in IAmong the 24 pTDP-43-positive ALS muscle samples, 10 were obtained from paraspinous musculature (41.7% of positive samples), 7 from diaphragm (29.2%), three from deltoid (12.5%), and one from gastrocnemius (4.2%). Three positive samples were from a muscle group not specified in the original autopsy report (12.5%) and were simply designated as “muscle, NOS.”P = 0.0092, OR = 4.25). No significant pTDP-43 and individual muscle group association (positive or negative) was seen for deltoid , quadriceps , or for paraspinous  or diaphragm  considered separately  . The combinations of pTDP-43-positive samples included: diaphragm and paraspinous (2 patients), diaphragm, paraspinous, and deltoid (1 patient), and diaphragm and deltoid (1 patient). Three of these patients had clinically-designated sALS (75%) and c9ALS was present in two of these four (50%).n = 19; 13 males, 6 females) had a median age of 64 years , median disease duration of 1114 days , and included three fALS and four c9ALS patients. Patients had limb (n = 11), bulbar (5), and multifocal (3) sites of symptom onset. The group without pTDP-43 pathology had a median age of 59.5 years , median disease duration of 1085 days , and included 7 fALS and 9 c9ALS patients. These pTDP-43-negative patients had limb (n = 27), bulbar (7), and multifocal (3) sites of symptom onset.The characteristics of ALS patients with and without pTDP-43-positive muscle samples are shown in Table P = 0.1) or disease duration . Likewise, no significant association was seen between pTDP-43 status in muscle and fALS status (OR = 0.78), c9ALS status (OR = 0.8), or site of symptom onset (OR = 0.57). Utilizing data from an earlier study [P = 0.67).No significant differences were identified between ALS patients with and without pTDP-43 muscle pathology with respect to age  included 9 females and 8 males with a median age of 60 years . Samples of non-ALS paraspinous muscle (n = 8) included 6 females and 2 males with a median age of 62 years . Among non-ALS samples, only IBM cases showed pTDP-43-immunoreactive inclusions . However, IBM and ALS patients with pTDP-43 inclusions did significantly differ in the spatial extent of pathology, with IBM patients having a median of 67% of LPFs involved .Four cases of IBM  were from three males and one female with a median age of 59 years. Among these samples, only IBM cases 100%) had pTDP-43 inclusions, which were also p62-immunoreactive . Five unstained sections, cut at 4 μm and labeled “ALS1–3” and “IBM1–2”, were shipped to the laboratory. Their results matched the spatial distribution and staining intensity of pTDP-43 in all IBM and ALS samples  of 0.54) (P = < 0.0001) in SQSTM1 and 1.67 fold increase in TARDBP (SEM = 0.31) (P = 0.0128). For IBM samples, relative to controls, there was a 1.33 fold increase in SQSTM1 expression (SEM of 0.26) (P = 0.012) and 1.31 fold increase in TARDBP expression (SEM of 0.29) (P = 0.014).As shown in Fig. 7This study demonstrates that aggregates of phosphorylated TDP-43 may be identified in the skeletal muscle of both sALS and fALS patients, including patients with and without c9ALS. This implicates axial skeletal muscle as an additional site of pTDP-43 pathology in ALS. A muscle group-specific difference in muscle pathology was also suggested by the finding that pTDP-43 inclusions were significantly more frequent in samples from axial muscle groups than appendicular groups , and up-regulation in gene expression (for TDP-43 and p62), suggests this may be an endogenous pathology in muscle that engages the autophagy pathway. Increased expression of p62 has also been seen in non-ALS, non-IBM muscle diseases, including genetically driven forms of muscular dystrophy [To our knowledge this is the first study to systematically demonstrate the presence of pTDP-43 aggregates in the myofibers of ALS patients, particularly in axial muscle groups. This implicates ALS muscle as an additional site of pTDP-43 pathology, as previously demonstrated in motor neurons, non-motor neurons, and glia. The downstream effects of cytoplasmic pTDP-43 pathology in ALS muscle cells requires further study, though studies in transgenic animals expressing mutant human TDP-43 have identified a toxic gain-of-function from cytoplasmic aggregation, leading to transcriptional dysregulation, including within histone processing genes . If cellystrophy , so it mAn alternative mechanism explaining pTDP-43 aggregates in ALS muscle is a “prion-like” transfer of pTDP-43 protein, possibly through anterograde axonal transport. This model of neuron-to-neuron spread has been proposed to explain dissemination of pTDP-43 pathology in brain and spinal cord . If tranOPTN) [UBQLN2), sequestosome 1 (SQSTM1) [VCP) [Our study also identified co-localization of p62/ sequestosome-1 in pTDP-43 inclusions of ALS muscle, which suggests the possibility that the autophagy pathway is activated. This recapitulates pathology well described in inclusion body myositis (IBM). Further comparison of ALS and IBM muscle samples may provide insight as to how pTDP-43 inclusions arise in vulnerable ALS muscle groups. To date, there have been few studies of pTDP-43 (or p62) pathology in ALS muscle. None of these, to our knowledge, have examined both axial and appendicular muscle groups or compared findings in ALS and IBM, and non-ALS, non-IBM samples with neurogenic atrophy. Previous studies do include a negative study of ALS quadriceps biopsies from 30 patients  (the 26 OPTN) , ubiquil(SQSTM1) , and ubi1) [VCP)  (the las1) [VCP) , in addi1) [VCP) . The finAn intriguing finding of this study was that pTDP-43 pathology was significantly enriched in axial muscle samples. It is known that physiologic abnormalities in paraspinous muscle by electromyography (EMG) are reliable in confirming the clinical diagnosis of ALS and help to distinguish the disease from its mimics . Paraspimus), but not nervous tissue, can lead to neurologic and pathologic ALS [SOD1 G93A mutation also show progressive muscular atrophy, mitochondrial dysfunction, and reduced muscle strength [LC3.Studies in the mSOD1 mouse model of ALS have provided direct evidence that cell autonomous pathology in muscle can drive the development of the disease . The musogic ALS  with ubistrength  and the TARDBP and SQSTM1 expression by real-time PCR supports this as a possible cell autonomous pathology in ALS, though future studies using unbiased techniques with a wide range of transcripts for normalization are needed . Additional studies utilizing tissue dedicated for electron microscopy study, with registration to histologic data, are required to clarify the ultrastructural features of the pTDP-43-positive material in ALS muscle. Nonetheless, our findings suggest that the impaired clearance of misfolded proteins in ALS, a well-recognized mechanism in motor neuron degeneration, may play an important role in pTDP-43 pathology in ALS muscle.In summary, this study implicates axial skeletal muscle (diaphragm and paraspinous muscles) as an additional site of pTDP-43 pathology in ALS. Up-regulation of Additional file 1:Figure S1. ALS muscle samples from three patients  with pTDP-43-immunoreactive inclusions. pTDP-43-stained sections (Proteintech clone 22309-1-AP) and H&E-stained sections from Houston Methodist (lab of MDC) are shown in the left three panels for each patient. The right-most panel of each patient shows repeat pTDP-43 staining at the University of Kentucky using the protocol of that laboratory and a different pTDP-43 antibody . All images are photographed at 400x without magnification except for five images photographed at 200x . Figure S2. Four muscle samples from a recent autopsy of an ALS patient not included in this study. The patient had a clinical diagnosis of C9ALS and a positive family history of ALS (fALS). Examination of brain and spinal cord confirmed ALS and showed characteristic p62-immunoreactive and TDP-43-negative lesions in brain, consistent with C9ALS. Examination of seven muscle samples using p62 and pTDP-43 immunohistochemistry showed unequivocal pTDP-43-reactive lesions in muscle fibers of thoracic paraspinous and diaphragm muscle, an equivocal focus in sacral paraspinous muscle (not shown), but no pTDP-43-positive lesions in cervical paraspinous muscle (not shown). The inset of diaphragm shows the same focus on a deeper histologic section, also stained for pTDP-43, further revealing the thread- and dash-like inclusions in this sample. All images are photographed are 200x except for diaphragm, which is photographed at 400x. Figure S3. 1% Thioflavin S staining was performed in Alzheimer’s disease (AD) control tissue from occipital cortex and hippocampus/ subiculum , IBM muscle , pTDP-43-positive ALS muscle samples with electron microscopy data shown in Fig 6 , all photographed at 600x magnification, and a pTDP-43-negative ALS muscle with atrophy and abundant lipofuscin pigment (ALS50) (600x). Green arrows highlight thioflavin S-reactive material in all samples, whereas white arrows indicate autofluorescent material, such as lipofuscin pigment , which appears yellow-orange in images combining FITC/ DAPI/ and TRITC filters (see Methods for detail on immunofluorescence microscopy). For ALS sample ALS43, the inset at lower left (600x) shows the similar shape of inclusions in this area by pTDP-43 immunohistochemistry. (PDF 1807 kb)