Our strategy suggests that the stripe patterned arrays of polymer films as sacrificial templates can be a simple route to creating highly sensitive biointerfaces and highlighting the development of new chemically patterned surfaces composed of graphene-based nanomaterials.Patterning of surfaces with a simple strategy provides insights into the functional interfaces by suitable modification of the surface by novel techniques. Especially, highly ordered structural topographies and chemical features from the wide range of interfaces have been considered as important characteristics to understand the complex relationship between the surface chemistries and biological systems. Here, we report a simple fabrication method to create patterned surfaces over large areas using evaporative self-assembly that is designed to produce a sacrificial template and lithographic etch masks of polymeric stripe patterns, ranging from micrometer to nanoscale. By facilitating a roll-on-plate geometry, the periodically patterned surface structures formed by repetitive slip-stick motions were thoroughly examined to be used for the deposition of the Au nanoparticles decorated graphene oxide  and the formation of conductive graphene channels. The fluorescently labeled thiol-modified DNA was applied on the patterned arrays of graphene oxide (GO)/AuNPs, and biotin-streptavidin sensitive devices built with graphene-based transistors (GFETs, effective mobility of ~320 cm Over the past decades, there has been considerable progress in the development of multifunctional electronic devices capable of detecting chemical and biological substances and measuring nanoscale phenomena . Recent In this context, among other nanomaterials, graphene, two-dimensional (2D) single-layer one-atom-thick graphitic carbon material has attracted widespread interest owing to its exceptional electronic, optical, chemical, thermal, and mechanical properties . Hence, Herein, we report a facile approach to fabricate highly aligned stripe mask patterns with lines and spacing configuration, using a simple upper moving roll on a flat substrate to confine the evaporative droplet of a polymer solution. After the drying of the polymer solution in the dynamic self-assembly, a so-called “coffee ring” pattern can be formed in gradient stripes . The effR = 1.33 cm, and the diameter, D = 1.5 cm) were used as a upper moving surface and flat substrate, respectively, constructing a unique confined geometry to trap a polymer solution, as schematically illustrated in C = 0.5, 0.25, and 0.125 mg mL−1). The dissolved PMMA solutions were filtered with a 0.2 μm hydrophobic membrane filter. Prior to use, Si substrate and cylindrical roll were cleaned with piranha solution (sulfuric acid: hydrogen peroxide = volume ratio of 7:3) imposing hydrophilicity on the surfaces, and then extensively rinsed with deionized water and dried by blowing N2 gas. The moving roll was controlled by the linear motorized translation stage fixed with both ends, and the cylindrical surface was in full contact with the lower stationary flat substrate . Prior to the close-contact between the roll and the flat substrate, 30 μL of PMMA solution was loaded and trapped following the roll and flat surface owing to capillary forces. Finally, the upper roll was traveled at an optimized speed of 7 μm s−1, leading to the spontaneous formation of the PMMA stripe patterns toward the opposite direction. These fabrication procedures modulated the dimensions of PMMA stripes with only the solution concentration  during the evaporation process controlled by a constant roll speed at ambient conditions (~24 °C).To produce the highly-ordered arrays of poly (methyl methacrylate) (PMMA) stripes, Si substrate and cylindrical tube  made from fused silica , 0.250 mL of an aqueous 0.01 M solution of gold chloride trihydrate  was added to 7.5 mL of a 0.10 M Hexadecyl-trimethylammonium bromide  solution. Subsequently, the mixed solution was processed by ultrasonication for 8 h at room temperature, and then 10 mL of the mixture was transferred to a Teflon-lined autoclave for the hydrothermal reaction at 180 °C for 12 h. The as-prepared hybrids were cooled naturally to room temperature.The exfoliated GO solution and the GO/AuNPs composite were synthesized by our previously reported method via the modified hydrothermal method . In a ty−1 with an optimized deposition number (100 cycles) to coat the surface of the Si substrate between PMMA stripe patterns. Finally, selective removal of the PMMA patterns was performed to lift off the GO-AuNPs that are deposited in the undesired region  using organic solvent. Then, the sample was dried by blowing N2 gas after thoroughly rinsed with deionized water and isopropyl alcohol (IPA). The GO/AuNPs solution was uniformly deposited on the gradient PMMA stripe patterns via the flow-enabled self-assembly (FESA) process, as described in our previous report ; the PMMThe patterned GO/AuNPs surface was then immobilized for 24 h with a drop of green fluorescent dye-labeled thiolated oligonucleotides deionized (DI) water solution -3-aminopropyl)]-propyl-2-O-succinoyl−longchain alkylamino-CPG; typically, 25 μL). In this process, the GO/AuNPs sample was sealed with a glass coverslip surrounded with polydimethylsiloxane (PDMS) gasket to prevent water evaporation. After the immobilization, the sample surface was extensively rinsed with DI water to wash away unbonded oligonucleotide. The thickness of oligonucleotide adsorbed on the sample surface was ~7–10 nm when measured by the atomic force microscope.2/Ar (20 sccm/50 sccm) flow from room temperature to 1050 °C at a constant pressure (100 mTorr). Then, the reaction gas mixture, CH4/H2/Ar (20/60/50 sccm), was introduced into the reaction tube for 20 min for the graphene growth. Finally, the quartz tube was cooled down at 3 °C s−1 until it reached room temperature. For the transfer process for the grown graphene on the Cu foil, PMMA solution  in toluene (10 wt%) was spin-coated onto the top of the graphene-grown Cu foil at 3000 rpm for 30 s. The Cu foil under the PMMA/graphene film was then fully etched in an optimized etchant . Finally, the graphene was transferred onto thermally grown SiO2  on a heavily doped p-type silicon wafer (resistivity of ~0.001–0.005 Ω∙cm), followed by the dissolving of the PMMA layer with acetone.A Cu foil was prepared for a conventional thermal CVD process . Firstly3 bonds with Si–OH groups, and then bonded with the BioGFET formed on SiO2/Si substrate  on a Si substrate ; the patubstrate  34]..3 bonds 2 gas. Separately, streptavidin was dissolved in the 0.01 M buffer solution ; the prepared protein solution was ready to be bound or conjugated on the biotinylated graphene channel surface of BioGFET in the microfluidic channel device for real-time detection.The graphene-based BioGFET substrate was sequentially modified with poly(ethylene imine) , poly(ethylene glycol) , and DI water solution at room temperature, using a drop-based immobilization or in microfluidic channels. After the PEI/PEG specific surface modification, the channel regions were exposed with a drop of 15 mM biotin-N-hydroxy-succinimide (NHS) ester (Sigma Aldrich) in dimethylformamide (DMF) solution, followed by incubation at room temperature for 12 h. Next, the surface-functionalized device was cleaned with DMF and DI water and dried by blowing NL), defined as the distance between source and drain, and channel width (W) were fixed at 7 μm and 50 μm, respectively; the graphene-based BioFET measurement was carried out using a semiconductor parameter analyzer (Agilent 4156A) in ambient condition.The samples were observed by optical microscopy (Olympus BX51 and IX81-F72) in reflective mode, scanning electron microscopy , and transmission electron microscopy . The surface of morphologies was collected using an atomic force microscopy (AFM) in non-contact mode (Park System XE-100). Raman spectra for GO and CVD grown graphene were obtained by Raman spectroscopy with 532 nm laser excitation . In addition, after patterning the graphene channel using gradient stripe patterns of PMMA etch-mask with a condition of the mild oxygen plasma etching , the source/drain electrodes were prepared by standard photolithography (AZ-GXR 601) and lift-off process to define patterns of Cr (5 nm)/Au (20 nm) electrodes on patterned graphene/Si substrate. The channel lengths (2/Si). In the experimental scheme, we designed a simple and viable route to spontaneously produce a set of highly ordered PMMA stripes. The first step involves the loading of a solution of PMMA in the restricted geometry, composed of an upper cylindrical roll on the lower receiving substrate placed on the stage. This approach is similar, but slightly different compared with our previous studies −. The electrostatic repulsion between the negatively charged exfoliated GO nanosheets and anionic Au precursors leads to the homogeneous GO-[AuCl4]− suspension for a period, and then the affinity of Au3+ with the GO nanosheets owing to the formation of the grafted hydroxy-Au compound through the replacement reaction between HAuCl4 precursors and hydroxyl groups attached to the defects of GO nanosheets; the absorption of Au3+ to GO surface can be proceeded by Galvanic displacement, which induces the deposition of AuNPs on the GO sheets in the aqueous solution by the autocatalytic reduction. This reduction of GO-[AuCl4]− could be promoted by adding the gallic acid  as a reducing agent and stabilizer; thereby, the Au3+ can be partially reduced and the subsequent absorption on the exfoliated GO sheets is triggered simultaneously. Raman spectroscopic analysis was also performed to investigate the structural characteristics of GO and GO/AuNPs sheets, as shown in −1 (D band) and ~1600 cm−1 (G band), where the D band arises from the vibration of carbon atoms with dangling bonds in-plane terminations of disordered graphite, and the G band originates from the in-plane stretching of ordered sp2-bonded carbon atoms in the hexagonal lattice. In the case of the as-prepared GO/AuNPs sheets, the enhanced intensity and red-shift of Raman spectrum with the increased ID/IG ratio of ~1.28 were observed, as compared with that of ~0.91 on the pristine GO sheets. These results indicate that the GO sheets were partially reduced by the combinatory reactions with the structural defects of GO sheets .To fully utilize the fabricated PMMA stripes, the template-assisted assembly of NP-modified GO nanosheets was performed as shown in 2/Si substrate were used as a sacrificial template to guide the formation of aligned arrays of GO/AuNPs stripes. When the aqueous GO/AuNPs solution (15 μL) was injected into the confined geometry of the upper blade on the PMMA stripe patterned substrate, the GO/AuNPs solution spread along with the different wetting in the restricted area, forming a meniscus by the capillary reaction (−1) with a preprogrammed number of cycles (~100), controlling the liquid thin layer of the GO/AuNPs solution. Specifically, the periodically alternating microstructure in hydrophobic PMMA stripes and hydrophilic SiO2 stripes  provided preferential wettabilities for the GO/AuNPs composites dispersed in DI water [2/Si stripes underneath of the receding waterfront, following the evaporation-induced capillary flow (X4 (C = 0.5 mg mL−1). As shown in X1) as presented in 2 stripes) defined by the patterned PMMA templates during the evaporative assembly process [DI water . As the ary flow . The dim process .C = 0.125 mg mL−1 at X4). By zooming in on a specific area  was constrained, and the upper cylindrical roll was slowly moved at a constant speed of 7 μm s−1 to form highly aligned arrays of PMMA stripes . As a result, the gradient PMMA stripe patterns successfully printed on the graphene surface after the complete assembly process  was performed to etch away the exposed graphene films . 2 surface; the white dotted line markedly indicates the boundary between the graphene and SiO2 region (uncovered by graphene). The aligned arrays of the stripe patterned graphene films were measured by SEM, as shown in 2/Si substrate; the careful alignment of the mask on the patterned graphene surface was required. In the structure, the highly-doped Si with a SiO2 (300 nm) was underlying as a gate with a SiO2 gate dielectric layer. In some other cases, we transfer the graphene sheet onto the priorly prepared electric pads, and then the patterning process was performed using the confined geometry. Surprisingly, the fabrication of GFET proceeds with low levels of defects and local disorder, thereby leaving the patterned graphene supported directly on the probing Au electrodes  on the SiO2/Si substrate, providing electrical isolation for each channel. To clarify the pattern formation of graphene on electrical pads on SiO2/Si substrate, the produced GFET was observed by an optical microscope, AFM, and SEM, as demonstrated in We further extended the present study to a smooth flat surface covered with CVD grown graphene toward the large scale etch-mask for the well-ordered graphene stripes by the self-assembly process based on the roll-on-plate geometry. The multichannel arrays of stripe patterned graphene being active in devices, formed with ultrathin patterned films, enables an improved current output, and may provide statistically beneficial device-to-device uniformity in properties even with the graphene films that are electronically heterogeneous . Figure  process b; differ−1), G (~1611 cm−1), and D (~1370 cm−1) peaks for monolayer graphene. In principle, the number of layers of graphene can be estimated by the ratio of 2D peak and G peak intensity to distinguish mono- and few-layer graphene. The intensity ratio of the G peak to the 2D peak  resulted from the patterned monolayer graphene, where I2D/IG was ~1.82, which also indicates the integrity of graphene was maintained regardless of the sequential patterning and etching process. Consequently, transfer characteristics of GFETs were measured by a semiconductor parameter analyzer under ambient conditions. Ids) as a function of gate voltage (Vg) at forwarding sweep between −100 and 100 V for a source/drain bias (Vds) of 0.05 V, and the characteristic output graph (Vg: −60 to 60 from the top) is shown in the inset. The device exhibits Dirac points in the positive Vg region (~2.8 V) and represents the p-type transistor behavior, and the current modulation ratio (Ion/Ioff) was ~2.5–3, which yielded the typical values from the micropatterned graphene FETs. This low level of on and off current ratio can be improved by the rational design of graphene  because the graphene bandgap can be open with lateral charge carrier confinement with suitable edge effects by narrowing the width of the graphene ribbons into nanoscale , as previously reported [reported ,54.μ, can be calculated from the transfer curve, using the measured peak transconductance and typical model. We define the effective field-effect mobility of the FET device asL and W are the channel length and width, respectively; oxC is the relative dielectric constant of the gate dielectric; and dsI, dsV, and gV are the drain-source currents, the drain-source voltage, and the gate voltage, respectively. Within the measured data set, resulting primarily from device-to-device variations in properties, the typical hole mobility ranged from ~270 to ~320 cm2 V−1 s−1 for the GFET devices. From this demonstration, PMMA etch mask utilized by the roll-on-flat geometry was a highly reliable method for device fabrication with its excellent ability by optimizing the graphene growth conditions and manipulation techniques, identifying the integrated GFET structures and property study [In addition, the linear-regime mobility, ty study .The prepared GFETs can be utilized as a biosensor platform  to construct active functional channels because it is a zero bandgap semiconductor, and the bandgap can be tuned by surface modifications using biomolecules such as biotin terminal groups that can serve as a binding site for streptavidin ,57. Ther2/Si substrate with areas that are comparable to the microfluidic dimensions of PDMS are observed. Thus, the design layouts represent both large sensing regions and ultrathin geometries in the structure for the biotin-streptavidin system, which is a standard model for protein–ligand interactions in our experiment scheme  as a function of gate voltage (gV) with forward sweep for a source-drain bias (dsV) of 0.05 V for a typical BioGFET; the channel length, L, and the width, W, were 10 and 100 μm, respectively. The gate voltage of the neutrality point for the transfer curve was located at around 0 V, and the p-channel mode was dominant in the device operation based on the bottom-gated transistors with less effect of oxygen adsorption [gm) in p- and n-channel mode of ~1 µS and ~0.4 µS, respectively. The inset graph represents a typical I–V curve of a BioGFET and shows a solid linear fit with linear Ohmic behavior. After the sequential modification of biotin/PEI/PEG on the active channels in the microfluidic system, the transfer characteristics were apparently changed into ambipolar transport behavior. Complemental interaction of biotin occurs through covalent binding to the primary NH2 group, thereby promoting the overall electron-donating function of PEI/PEG and leading to a device characteristic that is consistent with the removal of electrons from the device, as shown in n-channel mode appearing that exhibits a slight shift of the Dirac point to the negative gV region. As the PEI molecules were involved in binding to biotin, the p-channel mode of conductance observed before the chemical modification was not fully recovered, and the negatively charged biomolecules, captured on the receptors, produce an electrostatic gating effect, which is transduced into an obvious current drop. Prior to use of the BioGFET for the streptavidin detection, a control experiment was performed to demonstrate the electrical response on the biotinylated polymer-coated device to 0.01 M PBS buffer flow through a PDMS microfluidic channel  and biotin/PEI/PEG on the GFET (red), respectively. Finally, the real-time electrical responses on the BioGFET to streptavidin were performed as shown in n-channel mode , as observed in the graph. As extracted, the transconductances in the p- and n-channel were increased by ~6.7%  and decreased by ~33% , respectively; the n-channel mode of the transconductance shifted the notable magnitude in the operation, whereas the hole-conductance of the p-channel mode indicated small changes. This phenomenon might be attributed to the asymmetric doping effect in the graphene channels that was not exclusive to one dopant, but rather the result of more complicated transport mechanisms [n-doped graphene channels. The current response was larger with higher concentrations, and the response tended to peak at a concentration of 100 nM. The increased carrier reacting to the concentration of streptavidin at the nanomolar concentration caused sensitive changes in the BioGFET in highly reproducible ranges. Conclusively, the operating behavior on the graphene channels of BioGFETs is primarily owing to the consequent n-type doping effect of the biotin complex, potentially upon the binding of streptavidin detection in the microfluidic channel. With reasonable sensitivity in biotinylated BioGFETs as a biosensor, the multichannel graphene arrays were effectively rendered and successfully demonstrated for a single protein monitoring with the help of the microfluidic channel system [sorption , estimatchanisms ,63. Figul system .In summary, we have developed a simple, yet robust evaporative self-assembly strategy to spontaneously produce highly ordered PMMA stripes over a large area, facilitating a roll-on-plate geometry. This provides a unique environment to control the flow of PMMA solution by repetitive slip-stick motions with a limited evaporation rate between a moving cylindrical roll and a lower flat receiving substrate. The formation of stripe patterned arrays of PMMA was optimized by modulation of the solution concentration and the moving speed of the roll. To engineer the periodically patterned surface of PMMA stripes, they were served as an evaporative-guided template and lithographic etch masks as sacrificial patterned surfaces. Thus, highly ordered structural topographies and chemical features were beneficial to understand the relationship between the surface chemistries and biological systems. One example, the fluorescently labeled thiol-modified DNA, was immobilized on the arrays of GO/AuNPs by template-assisted self-assembly that may improve fluorescein behaviors by unique absorption and scattering properties for the biointerface. In addition, we demonstrated a lithographic approach to utilize the highly aligned arrays of PMMA stripes as etch masks for CVD grown graphene. With a simple surface modification process, such as co-functionalization with biotin/PEI/PEG on the conductive multichannel of graphene, molecular sensitive BioGFETs could be built to detect streptavidin with the help of the microfluidic channel system. Our presented study suggests that the stripe patterned arrays of polymer films as sacrificial templates can be a simple route to creating amenable biointerfaces and chemically patterned surfaces synergistically combined with the development of a new class of nanomaterials such as graphene and other 2D nanomaterials for a wide range of biosensor platform ,66,67.


We read with great interest the study by Baessler et al. , which cThe study showed that risk communication topics were only mentioned in 6.1% of PBL sessions compared to 34.8% of practical training sessions. Likewise, demonstration material used for risk communication was only used once in PBL sessions. Having been involved in such sessions, we have also noted the lack of teaching in risk communication. The use of PBL allows understanding and application of important medical concepts and, as medical students at a university adopting a PBL curriculum, we believe that PBL becomes particularly effective in appreciating more demanding and niche topics such as risk communication. Therefore, we wonder why more medical schools do not incorporate learning opportunities for risk communication. Whilst integrating risk communication into PBL does pose challenges, such as recruiting tutors with both clinical and statistical expertise and developing specific patient cases, we can learn from the various models employed by medical schools in Australia, which integrates statistical material into PBL sessions [The authors make note of the fact that few questions were asked in sessions where risk communication was taught, indicating low interest in the subject amongst students and a ‘failure to induce discussion. A study conducted in the University of Makassar, Indonesia, delved into factors influencing questioning powers of students in class and found that, although interest in materials did affect students’ likelihood to ask questions, other factors such as personality characteristics of teachers, emotional state of the student and, particularly, being afraid of asking questions also contributed. Indeed, 72% of students felt afraid to ask questions in class, with many of them preferring to ask their friends instead or teachers face-to-face after class . As mediDespite the contributions of the paper, we do question the need for statistical values to be taught in-depth repetitively. The research found ‘in 90% of these teaching sessions 16 out of 19 common statistical values … were only mentioned without further elaboration’. Whilst we appreciate that statistical values are important, the authors have noted correctly student engagement is low in these sessions. Interestingly, the authors have found ‘that traditional literature handouts were the most preferred choice of supplementary material’. We, therefore, suggest that a literature handout with explanations of the 19 common statistical values could be produced and given to aid students in these sessions, not only increasing engagement but also providing standardised teaching to reduce the levels of ‘statistical illiteracy’.Academic staff could also observe lectures adding a different perspective to the delivery of lecture contents. This comparison could explain potential omissions, as seen with the 112 sessions where students ‘could not observe any risk communication teaching’, shedding light on positive avenues for reform in medical universities. Furthermore, we suggest that future studies should allow students to add free-text comments to evaluate risk communication teaching, as openly discussing the syllabus would be hugely beneficial in student understanding. A study carried out in South Africa found that qualitative course evaluations were a suitable alternative to quantitative surveys . A more


Extensive research on functionalized graphene, graphene oxide, and carbon nanotube based cement composites has been carried out to strengthen and overcome the shortcomings of construction materials. However, less literature is available on the pure graphene based cement composite. In this review paper, an in-depth study on a graphene-based cement composite was performed. Various structural forms of graphene and classifications of graphene-based nanomaterial have been presented. The dispersion mechanism and techniques, which are important for effective utilization in the construction industry, are reviewed critically. Micro-scale characterization of carbon-based cement composite using thermogravimetric analysis (TGA), infrared (IR) spectroscopic analysis, x-ray diffractometric (XRD) analysis, and morphological analysis has also been reviewed. As per the authors’ knowledge, for the first time, a review of flow, energy harvesting, thermoelectrical, and self-sensing properties of graphene and its derivatives as the bases of cement composite are presented. The self-sensing properties of the composite material are reported by exploring physical applications by reinforcing graphene nanoplatelets (GNPs) into concrete beams. The construction industry makes a significant contribution to economic growth in every part of the world. Every year, 20–35 billion tons of concrete is used globally, making it the most widely used construction material , and itsThe literature on the review of nanotechnology in concrete  has highQureshi and Panesar  in theirIn this review article, a detailed study on the graphene-based cement composite is performed. A brief explanation of graphene nanostructures is presented in Graphene is a single layer carbon sheet and one of the most promising nanofiller used to enhance cementitious materials ,32. PracThis section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation as well as the experimental conclusions that can be drawn.These newly developed engineered nanomaterials are characterised by their morphology: zero-dimensional (0D) nanoparticles , i.e., carbon black; one-dimensional (1D) fibers (straight and high aspect ratio), i.e., carbon nanotubes; and two-dimensional (2D) sheets, i.e., graphene and GO . Figure Molecules, which consist of wrapped graphene by means of the introduction of pentagons on the hexagonal lattice are called zero dimensional nanoparticles. These allotropes of carbon were discovered by Kroto et al. . They ar2/g and aspect ratio of more than 1000, due to varying length of carbon nanotubes [Compared to zero-dimensional nanoparticles, spherical shape one-dimensional nanofibers have a high aspect ratio i.e., carbon nanotubes. Exfoliated GNP (xGnP) and carbon nanotubes (CNT) share the same chemical structure . CNTs aranotubes . CNTs haanotubes . Konsta anotubes  noted 25anotubes , when 0.anotubes . That isanotubes . Severalanotubes ,51 notedanotubes  states tIn contrast to CNTs, graphene and GO are the two-dimension sheet-like structures and have a considerable surface area. GO has a thickness of a single atomic layer while having lateral thickness reaching to tens of micrometres, which provides large surface area and immense aspect ratio . It has In 2004, Novoselov et al.  derived 2 g−1) [−1K−1) [Recently, the 2D flat graphene sheet has gained an enormous attention in science for its promising and outstanding properties: high intrinsic strength (130 GPa) , large s2 g−1) , high th [−1K−1) ,63 and f [−1K−1) ,65,66. T [−1K−1)  mentione [−1K−1)  also fou [−1K−1) .Dispersion is a primary problem related to fabricating cementitious nanocomposite because Van der Waals force forms an agglomeration of the nanoparticles . DispersThe following section is regarding the application of dispersant to the Graphene and its derivatives. As graphene flakes are hydrophobic and tend to form coagulation in the aqueous solution, therefore, numerous researchers used different dispersants to obtain a stable suspension. Stankovich et al.  used redSixuan  used acet-octylphenol decaethylene glycol ether , and Dispex N40. They concluded that most favourable dispersant was Triton X-100, a surfactant molecule, as it showed the highest absorption peak in the UV-vis absorption spectra, as presented in Sharif et al.  examinedRecently, Silva et al.  used isoThe dispersion mechanism for dispersant and graphite nanoplatelets was explained by Sixuan . The autAnother way to deal with dispersion problem with graphene and its derivatives to apply sonication. The sonication is the act of agitation of particles by means of applying energy. The ultrasonication is referred to as the waves having a frequency of more than 20 KHz. The ultrasonic electric generator takes the biggest part of sonication device and generates a signal, normally about 20 KHz, which charges a transducer and it transforms the electric signal to mechanical vibrations. These vibrations are further augmented by the sonicator and transmitted to the probe, which transfers the vibrations to the solution. The quick movement of the probe produces a cavitation event. It takes place when the vibrations generate multiple microscopic bubbles in the solution, some wedged intermolecular space is developed and breaks down continuously under the influence of the weight of the solution. Constant generation and breakdown of thousands of these bubbles develop the robust waves of vibration, which pass through the solution and crush the particles . The eneMehrali et al.  applied Ultraviolet-visible spectroscopy (UV-vis) is defined as an absorption or reflectance spectroscopy in the UV spectral region. It is also known as a competent method to examine the dispersion of graphene structure, particularly, the GNPs and CNTs. Jiang et al.  used theTherefore, it can be concluded from the literature of the dispersion section that the ultra-sonication technique provides uniform dispersion to graphene flakes. The use of dispersant, i.e., polycarboxylate based high range water reducing admixture will help in exfoliation of graphene flakes. Additionally, the UV-vis spectroscopy method is extensively applied to monitor and assess the dispersion of nanomaterials in aqueous solution.Characterization refers to the procedures by which the material’s properties and structure are explored and measured. The graphene-based nanomaterials interact with hydrated cement products and influence the hardened properties of the composite material. Thus, it is important to study the micro-scale characterization of graphene-based cement composite. In this section, the characteristics of graphene-based cement composite are explored by (a) thermogravimetric analysis (TGA), (b) infrared spectroscopic analysis, (c) X-ray diffractometric (XRD) analysis, and (d) morphological analysis.Thermal analysis is a method which estimates the change in the materials properties depending on the temperature . The effWang et al.  observed−1 (sp2 hybridized C=C), 2918 cm−1 and 2850 cm−1 (symmetric and asymmetric stretching vibration of –CH2), pointing to the sp2 network [−1); (b) the sulphate region (1100–1150 cm−1); and (c) the material region (<1000 cm−1). Spectral data vary by the incorporation of different nanomaterials and assessments of these transitions will yield useful information. Shifting of these bands implies the stronger bonding, formation of hydrated products and polymerization in hydrated products [Vibrational spectroscopy is an approach for the assessment of the molecular structure. It provides useful information about possible chemical and physical interaction . FTIR sp network . Accordi network , FTIR spproducts ,113 whil−1 and 1118 cm−1 corresponds to C=O stretching of carboxylic acid and C-OH stretch of hydroxyl. These peaks confirmed that various oxygen-containing groups are attached to the surface of CNTs. When these surface treated CNTs were added to cement paste, a chemical interaction took place, as shown in −1 by 22 cm−1 indicated the probable existence of carboxylate while the disappearance of peak at 3643 cm−1 specified the chemical synergy between hydrated cement products with oxygen groups of carboxylic acid attached with the CNTS. In addition, the variation in spectral shape in the CSH region indicates the variation in CSH phases due to the functionalization of carbon nanotubes.Geng et al.  employed2O3, and n-SiO2 to cement composite was investigated by Murugan et al. [−1 was attributed to O–H bend, ettringite peak at ~1118 cm−1 was due to S–O stretch, silicates peaks at ~950 cm−1 was due to Si–O asymmetric stretch. Calcite peaks, which are indicative of carbonation, were recorded in all mixes at ~1414 cm−1 and ~874 cm−1, attributed to C–O stretching and C–O bend vibration respectively.The effect of rGO, n-Aln et al. . Figure 3S) was also found to be lower in graphene cement composite. In addition to this, Murugan et al. [X-ray diffractometer was broadly adapted for determining the crystalline structure of materials . Wang etn et al.  found th3S and C2S were reduced significantly by incorporating the graphene in the cement mix. Brittleness of composite mix was found less as compared with the plain sample because of the absence of Aft peak. It was suggested that the relative decrement of the C4AF  peak in the composite mix indicates the higher hydration rates due to nanocomposites.The XRD patterns for control mix, 0.125 PGO-CNC, and 0.125 GONO-CNC after 90 days of curing were obtained by Sharma and Kothiyal  and presVarious researchers have used field emission scanning electron microscopy (FESEM) to evaluate the morphology of cement-based composite prepared with graphene and its derivatives. The microstructure and pattern of hydrated crystals of graphene oxide based cement composite were investigated by Shenghua et al. . The SEMCui et al.  studied Cao et al.  stated tTherefore, it can be concluded form a literature of microscale characterization that no chemical interaction and new phase formation was observed in graphene cement composite. However, graphene-based nanomaterials act as accelerator and dense formation of hydrated cement product was found near the nanomaterials. A detailed and in-depth study is still required to completely understand and explore the influence of graphene-based nanomaterials on hydrated cement products at the microscale level.Concrete possesses great advantages which makes it the most extensively used building material . During Many researchers examined the influence of different nanomaterials on cement paste flow properties ,127,128.Yahia et al.  found thThe rheological properties of fresh cement paste with different surface area of GNPs, resting time and shear rate cycles were examined by Rehman et al. ,131 usinIt can be concluded from the existing available literature that new mathematical models need to be developed which will predict the rheological properties of nanomaterial-based cement composite. Moreover, the influence of graphene-based nanomaterials requires further exploration so that it can be successfully used in 3D printing applications of the construction industry.Consumption of cementitious building materials i.e., concrete is increasing due to its various advantageous characteristics such as high strength, durability, and resistance to fire . Sixuan Enhanced mechanical properties of graphene cement composite are attributed mainly to the bonding of graphene and its functional groups with CSH gel . Hou et 2σT/κ), where S is Seebeck coefficient in µVK−1, σ is electrical conductivity in Scm−1, T temperature in K and thermal conductivity κ in Wm−1K−1 respectively. In addition, ZT ≥ 1 is recommended for energy harvesting application purposes in buildings. Moreover, graphene has the capability to transform the non-conductive material into conductive material. According to Balandin [Energy harvesting is a concept through which existing ambient energy in the environment e.g., solar, wind, hydro energy has been converted into useful forms i.e., electrical energy . AccordiBalandin , graphen−4 was noted for 15% addition of graphite in cement composite at 75 °C. Overall, ZT values were found to be smaller due to lower electrical conductivity and seebeck coefficient of graphite cement composite. However, due to large areas of graphite cement composite in pavements and roofs being exposed to solar radiation, the solar energy can be converted into electrical energy during the summer season around the globe. It was Wei et al. , who for−1 for 15% GNP inclusion at 70 °C, while electrical conductivity of 16.2 Scm−1 and power factor of 1.6 µWm−1K−2 for 20% GNP inclusion as shown in −1K−1 was found for 10% GNP cement composite while 20% graphene cement composite showed highest thermal diffusivity as compared to other percentages of graphene cement composites. Moreover, the highest ZT of 0.44 × 10−3 was found for the 15% GNP cement composite at 70 °C. According to authors, the higher amount of GNP in the cement composite was beneficial for establishing the conductive network. It was concluded that graphene based cement composite significantly contribute to energy harvesting application in addition to improving the quality of indoor environment of buildings. With this application, graphene cement composite can harvest the energy, reduce electric consumption, and provide a substantial financial benefit [Ghosh et al.  performe benefit .Nanomaterials are gifted with the amazing characteristics and their incorporation in cement composite improved the properties of nanomaterial based cement composite. The reason for the presence of electrical properties of graphene is the half-filled band that permits free-movement of electrons. The π-bonds hybridize together to form the π-band and π*-band, which are responsible for the electrical properties of graphene . Self-seThe electrical properties of the cement composites are also of great importance and may be used to control damage in a concrete structure. The cement-based composite reinforced with conducting fillers can recognise its own strain by indicating the variations in the electrical resistivity values. According to Rehman et al. , the pieS1, S2, and S3 are the spacing in cm and calculated from current carrying probe to the voltage measuring probe. Various researchers used the piezoresistive characteristics of nanomaterials based cement composite for self-sensing prose [Han et al.  reportedng prose ,176,177.According to Hui et al. , the selXun and Kwon  studied The influence of different amounts of GNPs on the sensing behaviour of the cementitious composite was investigated by Hingiian and Pang . The autRehman et al.  investigAs existing well-known methods of health monitoring have some limitations in the application  and mostIn the literature, various nomenclature has been used which could create confusion for the readers. Therefore, a detailed description of graphene and its derivation including its precursors is provided in It has been noted that GNPs enhance the hydrated cement products at an early age of 7-days in contrast to 28-days. Furthermore, no chemical interaction and new phase formation in hydrated cement product took place in the production of GNPs and hydrated cement products. However, from infrared spectral analysis Li et al.  found thIt was found that the graphene based nanomaterials have several issues which, needs to be resolved before its usage in the construction industry. The following research gaps have been identified.(1)Very limited research has been found regarding the manufacture of nano-size cement particles and nano-binders however, it has great potential for the development of novel admixtures, nanoparticles, and nano-reinforcements.(2)Most of the researches on the dispersion of graphene and its derivatives were focused on surface modification, functionalization, and oxidation process. However, these processes damaged the atomic structure of graphene. Therefore, other methods are indispensably required for the dispersion of GNDs and preserving the atomic structure of graphene.(3)The enhancement mechanism in properties is not completely described as yet. Further research is required to study the regulating mechanism of graphene and its derivatives on hydrated cement crystals.(4)Flow properties of graphene cement composite and its dependence on various factors are missing in the existing literature. The variation of geometric flow with time, dispersing agent, shear rate and various types of graphene sheets is required to explore the flow of cement paste in the plastic state. Moreover, the best optimized rheological mathematical model needs to be sorted out, as a single rheological model cannot predict the flow behaviour of cement paste accurately. Thus, graphene cement composite demands further exploration to achieve maximum benefit from graphene.(5)The complete mechanism for accelerating the hydration reactions and enhancing overall mechanical properties has not been explained. Prominent differences in mechanical properties were found in experimental work conducted by various researchers under similar experimental conditions.(6)The distance between the current and voltage poles is required to be optimized for four-probe method. The potential application of graphene-based cement composite as an embedded smart sensor in the real concrete structure was seldom found in the literature. Moreover, the suitability, industrial demand, and compatibility of graphene cement smart sensors with the existing non-destructive health monitoring methods need to be determined.In this review paper, we have critically reviewed recent research on graphene based cement composites. It was demonstrated that dispersion of graphene is critical for its effective utilization in cement based composites. Several characterization techniques, such as TGA, IR, XRD, and SEM, were discussed in detail to evaluate the impact of graphene on the performance of cement-based composites. Conclusively, graphene was found to enhance the rheological, mechanical, thermoelectrical, piezoresistive, self-sensing, and damage memorizing capabilities of cement-based composites.


This paper is a review of the literature on the clinical role of oncomarkers in idiopathic pulmonary fibrosis (IPF) progression, and a description of the routine oncomarker trend in IPF patients over the longest follow-up yet reported. This is the first meta-analysis to review the results of studies evaluating the predictive prognostic value of circulating oncomarkers  for IPF. The study focused on the discovery of multiple biomarker signatures, such as combinations of oncomarkers, that are widely and routinely available in biochemistry laboratories. The combination of clinical parameters and biological markers could help achieve more accurate results regarding prognosis and response to treatment in IPF. Our results could pave the way for a more “personalized” medical approach to patients affected by IPF.p < 0.05). Ca15.3 concentrations were higher in the IPF, than the non-IPF, group at t3 (p = 0.0080) and t4 (p = 0.0168). To improve the specificity and sensitivity of Ca15.3, a panel of biomarkers was analyzed, with the IPF group as dependent variable, and chitotriosidase, Cyfra 21.1, Ca15.3, Ca125, and Ca19.9 as independent variables. Conclusions: This study focused on the discovery of multiple biomarker signatures, such as combinations of oncomarkers, that are widely and routinely available in biochemistry laboratories. The combination of clinical parameters and biological markers could help achieve more accurate results regarding prognosis and response to treatment in IPF. Our results could pave the way for a more “personalized” medical approach to patients affected by IPF.Background: Idiopathic pulmonary fibrosis (IPF) is a severe progressive interstitial lung disease. At 5-year follow-up, 15% of IPF patients develop lung cancer, which significantly reduces the survival rate. Here we review the literature on the clinical role of oncomarkers in IPF progression, and describe the trend of routine oncomarkers in IPF patients over the longest follow-up yet reported. Materials and methods: A systematic search of the literature in PubMed was performed to find relevant studies published up to 24 September 2020. The most common oncomarkers were chosen to select papers related to pulmonary fibrosis. Then, 24 IPF patients and 25 non-IPF patients, followed at Careggi ILD Referral Centre and Siena Regional Referral Centre for ILD, were enrolled consecutively. Results: A few studies reported an association between serum oncomarkers and severity of IPF. NSE, CEA, Ca19.9, and Ca125 were higher in the IPF, than in the non-IPF, group at every follow-up ( Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fibrosing interstitial pneumonia of unknown cause, occurring primarily in older adults, and limited to the lungs . Recent 2 = 92.3%, p < 0.001, mean CEA concentrations (IPF vs. non-IPF): 5.35 vs. 4.89 ng/mL; I2 = 91.9%, p < 0.001, mean Ca125 concentrations (IPF vs. non-IPF): 34.68 vs. 32.09 U/mL) (Higher concentrations of carcinoembryonic antigen (CEA) and Ca-125 were recorded in IPF patients with lung cancer, than in non-IPF patients (I09 U/mL) a,b.2 = 88.8%, p = 0.001, mean (IPF vs. non-IPF): 91.02 vs. 16.3 U/mL), Ca19-9 : 54.71 vs. 15.29 U/mL) and KL-6 : 1164 vs. 317 U/mL) were associated with disease progression in IPF patients (Serum concentrations of Ca15.3 (Ipatients a–c. In pp = 0.0070). In the IPF group, mMRC score at t0 differed from those at subsequent follow-up times (p = 0.0001). At 18-month follow-up (t3), three IPF patients had died, while no patient had died in the non-IPF group. Stratifying the study population according to therapy with pirfenidone or nintedanib, we did not observe any statistically significant difference of oncomarker concentrations or functional disease progression.The main characteristics of our population are reported in Statistical analysis was performed comparing each sampling time for each group ; moreover, a comparison analysis was performed between the two subgroups .p < 0.05) [Serum concentrations a,b of ch < 0.05) ,25,26,27p = 0.0294) and t4 (p = 0.0019) and the difference was statistically significant between t1 and t4 (p = 0.0327). Comparing oncomarker concentrations in the two groups, neuron specific enolase (NSE), CEA, Ca19.9 and Ca125 were higher in IPF patients than in the non-IPF group at every follow-up (p < 0.05). Ca15.3 concentrations were higher in the IPF than the non-IPF group at t3 (p = 0.0080) and t4 (p = 0.0168).The non-IPF group showed lower CEA concentrations at t0 than at t3 (p = 0.0369), t0–t4 (p = 0.0142), t1–t3 (p = 0.0350), and t2–t4 (p = 0.043).In IPF group patients, serum concentrations of Ca15.3 showed a statistically significant increase in the intervals t0–t3  (p < 0.0001) (p < 0.0001) (p < 0.0001) (p < 0.0001)  obtained by logistic regression was 88%  . With th 0.0001) , at t2,  0.0001) , at t3,  0.0001)  and at t 0.0001) . With rep < 0.01) at t3 with respect to t2 and t0 (p < 0.01). No significant differences (p > 0.01) in lung function parameters were observed in non-IPF patients in the serial follow-up.Regarding lung function , FVC%, fp < 0.05). Due to the limited statistical sample, no correlations between serological biomarkers and survival data could be detected. Correlation analysis between serum biomarkers and lung function parameters in the two groups are shown in The trend of functional parameters in the IPF population showed a progressive statistically significant decline at t3 and t4  for IPF. Although oncomarker concentrations were higher in IPF than non-IPF patients, no data were available on IPF progression, including mortality. A few studies have found higher levels of circulating oncomarkers in IPF than in non-IPF patients ,18,22. TA few studies have reported an association between serum oncomarkers and severity of IPF, and only two papers have evaluated the association with survival of these patients. These papers reported that elevated CEA and Ca125 concentrations were associated with increased risk of lung cancer in IPF patients with similar cut-off values ,16. KodaOne study showed a significant negative correlation of serum KL-6 levels with FEV1 and FVC percentage . The preIn the present study, we evaluated serial changes in chitotriosidase and circulating oncomarkers in a cohort of ILD patients divided into IPF and non-IPF groups. Increased chitotriosidase values have been repeatedly reported in sarcoidosis patients, and may predict clinical course and potential relapse of the disease ,26,27,28The clinical course of IPF is variable, ranging from slow progression over many years, to acute exacerbation and rapid loss of lung function. This, associated with a lack of biomarkers to predict disease progression and response to treatment, makes the clinical management of IPF very challenging ,33,34,35Significant correlations between oncomarkers and several functional parameters were found. In particular, CEA showed an indirect correlation with FEV1%, FVC%, and DLCO% decline at 18- and 24-month follow-up in IPF patients with respect to non-IPF patients. Our findings were in line with those of a prospective study by Fahim et al., who demonstrated elevated serum concentrations of CEA in IPF patients, and a significant negative correlation of the latter with lung function parameters . MoreoveOur results showed that CEA had the greatest sensitivity and specificity in distinguishing IPF from non-IPF at all follow-up times. Ca15.3 had a low sensitivity, but was the only marker to show a rising trend in IPF. However, a panel consisting of chitotriosidase, Cyfra 21.1, Ca15.3, Ca125, and Ca19.9 showed better sensitivity and specificity at all follow-up times than any single biomarker in distinguishing IPF from non-IPF patients. Histological assessment of Ca19.9 and Ca125 suggested that these proteins were markers of epithelial damage . In the Our data confirms the previous findings of higher serum levels of oncomarkers  in IPF than in non-IPF patients. Ca15.3 was correlated with poor prognosis. Interestingly, all except CEA decreased significantly after lung transplant [The limitations of the present study were the relatively small number of studies found for the meta-analysis, which did not enable us to explore the sources of heterogeneities.A systematic search of the literature in PubMed was performed to find relevant studies published before 24 September 2020, and limited to the following publication types , species (human), and languages (English). The most common oncomarkers  were chosen in order to select papers related to pulmonary fibrosis. The following medical subject headings were searched in (Title/Abstract): (“Ca-15-3” or “Ca15.3” “pulmonary fibrosis”), (“Ca19.9” “pulmonary fibrosis”), (“Ca125” “pulmonary fibrosis”), (“CEA” “pulmonary fibrosis”), , (“prostate specific antigen” “pulmonary fibrosis”), , (“chromogranin A” “pulmonary fibrosis”), (“neuron specific enolase” or “NSE” “pulmonary fibrosis”), (“Cyfra 21-1” “pulmonary fibrosis”), (“human chorionic gonadotropin” “pulmonary fibrosis”), , (“thyroglobulin” “pulmonary fibrosis”), (“Krebs von den Lungen-6” or “KL-6” or “MUC1” “pulmonary fibrosis”).Studies were included in the meta-analysis when they met the following criteria: studies focused on patients diagnosed with IPF according to the results of chest radiography, conventional thoracic computed tomography, HRCT, and pulmonary function tests (either FVC% or DLCO% < 80%); oncomarkers identified in serum; homogeneous units of measurement and methods of detection of oncomarkers; studies that provided sufficient data to complete cross-tabulations (2 × 2 tables) for evaluating the diagnostic accuracy of serum oncomarkers in IPF versus non-IPF patients. When the same population was published in different reports, the most recent or complete report was included. Case reports, case series, reviews, letters and conference abstracts were excluded due to limited assessment or analysis of data. Studies with insufficient data  for completing cross-tabulations were excluded as well.Data was extracted from eligible studies and summarized independently by two researchers (MD and LB). Any disagreement was resolved by consensus. The following information was collected from each study: first author’s name, year of publication, disease subtypes, affiliation of study, ethnicity of population, assay method, cut-off value, sample size, number of case groups, number of control groups. From cross-sectional studies we extracted mean concentrations and standard deviations (SD) of circulating oncomarkers in patients with and without IPF.2, respectively. When heterogeneity was high , random effects models were used as a pooling method.These data were processed with Covidence, Jamovi, and GraphPad Prism 9 software. For cross-sectional studies, the standard mean difference (SMD) and 95% CI were computed as the effect size of following comparisons: IPF vs. non-IPF. Heterogeneity between studies and the amount of variation derived from heterogeneity were evaluated by Q test and ISeventy-nine ILD patients followed at Careggi Interstitial Lung Diseases Referral Centre and Siena Regional Referral Centre for ILD were enrolled consecutively. Twenty-four diagnosed with idiopathic pulmonary fibrosis (IPF) and 25 non-IPF patients diagnosed with nonspecific interstitial pneumonia (NSIP) (n = 9), sarcoidosis (n = 8), pleuroparenchymal fibroelastosis (PPFE) (n = 2), respiratory bronchiolitis associated with ILD (RB-ILD), subacute hypersensitivity pneumonitis (HP) (n = 4), and cryptogenic organizing pneumonia (COP) (n = 2) were selected for consecutive serial evaluation of oncomarkers. We excluded IPF patients with a follow-up inferior to 24 months, those patients with concomitant infection, malignancy and acute exacerbation compliance, or those patients with non-definite usual interstitial pneumonia (UIP) pattern at CT scan. All the diagnoses were confirmed by multidisciplinary discussion, according to international guidelines (ATS/ERS).Of this selected population, 15 IPF patients (62.5%) were treated with pirfenidone and nine (37.5%) with nintedanib. Non-IPF patients were treated with corticosteroids  and immunosuppressant therapy  . All patients were naïve for therapies at the time of diagnosis (t0), and started nintedanib or pirfenidone treatments according to Italian national drug inclusion criteria.Medical history, physical examination, age, sex, ethnicity, BMI, vital signs, onset of symptoms, hospitalizations, concomitant diseases, smoking history, occupational history, current and previous therapy, blood gas analysis, and 6-minute walking test were recorded in a database. All patients gave written informed consent to participation in the study, which was approved by the local ethics committee CEAVSE .Lung function tests were performed according to ATS/ERS recommendations using a plethysmograph with corrections for temperature and barometric pressure and the parameters were expressed as % of predicted value: forced expiratory volume in 1 s (FEV1%), (FVC%), (DLCO%) by the single-breath method, and total lung capacity (TLC%). A six-minute walking test was performed according to international recommendations. These measurements were obtained from all patients able to perform lung function tests.Serum samples for assay of the following proteins/oncomarkers were drawn at baseline t0 and every 6 months of follow-up (6 months (t1), 12 months (t2), 18 months (t3), and 24 months (t4)): chitotriosidase (chitinase-1), carcinoembryonic antigen (CEA), cancer antigen 15-5 (Ca15-3), neuron-specific enolase (NSE), cancer antigen 19-9 (Ca19-9), cytokeratin fragment 21-1 (Cyfra 21.1), and cancer antigen 125 (Ca-125). Oncomarkers and chitotriosidase were assayed as previously reported ,39,42,43Data was expressed as mean ± standard deviation or median and interquartile range. Non-parametrical tests were adopted for data analysis: Kruskal–Wallis and Dunn´s multiple tests to compare the two groups (IPF and non-IPF) and to compare the sampling times each other . The Chi-squared test was used for categorical variables, as appropriate. The oncomarkers analyzed in this study were commonly used in clinical practice and the cut-off values were standardized: CEA > 2.5 ng/mL , Ca15-3 > 35 UI/mL (n.v. 0 e 32.4 U/mL), NSE > 15 (n.v. 0 to 15 ng/mL), Ca19-9 > 40 U/mL (n.v. 0–40 U/mL), Cyfra 21.1 > 3.5 mcg/mL (n.v. 0 to 3.5 mcg/mL), and Ca125 > 35 UI/mL (n.v. 0–35 UI/mL).Serum biomarker concentrations were also compared between groups, assessing areas under (AUC) the receiver operating characteristic curves (ROC). Logistic regression analysis, using the IPF group as dependent variable against non-IPF patients, to assess the potential of serum markers in discriminating the two groups at each sampling time, was adopted. Sensitivity, specificity, and positive and negative predicted values  were calculated for cut-off of the different variables. The Spearman test was used to look for correlations between variables. A p value less than 0.05 was considered statistically significant. All the statistical analysis and the related figures were carried out using GraphPad Prism 8.4 software.In conclusion, the study focused on the discovery of multiple biomarker signatures, such as combinations of oncomarkers, that are widely and routinely available in biochemistry laboratories. The combination of clinical parameters and biological markers could help achieve more accurate results regarding prognosis and response to treatment in IPF. Our results could pave the way for a more “personalized” medical approach to patients affected by IPF.Biomarkers are relevant in medicine, particularly in the realm of “personalized medicine”. Disease severity biomarkers are helpful tools, and they are valuable for predicting prognosis. It is known that every patient can have a different response to a specific therapeutic approach according to several immunological features. Our results could pave the way for a more “personalized” medical approach to patients affected by IPF monitoring oncobiomarkers.


NTRK splice variants in normal and neoplastic brain provides an intersection of these two rapidly evolving fields. Tropomyosin receptor kinase B (TrkB), encoded NTRK2, is known for critical roles in neuronal survival, differentiation, molecular properties associated with memory, and exhibits intricate splicing patterns and post-translational modifications. Here, we show a role for a truncated NTRK2 splice variant, TrkB.T1, in human glioma. TrkB.T1 enhances PDGF-driven gliomas in vivo, augments PDGF-induced Akt and STAT3 signaling in vitro, while next generation sequencing broadly implicates TrkB.T1 in the PI3K signaling cascades in a ligand-independent fashion. These TrkB.T1 findings highlight the importance of expanding upon whole gene and gene fusion analyses to include splice variants in basic and translational neuro-oncology research.Independent scientific achievements have led to the discovery of aberrant splicing patterns in oncogenesis, while more recent advances have uncovered novel gene fusions involving neurotrophic tyrosine receptor kinases (NTRKs) in gliomas. The exploration of  NTRK2) gene, exhibits intricate splicing patterns and post-translational modifications. Here, the authors perform whole gene and transcript-level analyses and report the TrkB.T1 splice variant enhances PDGF-driven gliomas in vivo and augments PI3K signaling cascades in vitro.Tropomyosin receptor kinase B (TrkB), encoded by the neurotrophic tyrosine receptor kinase 2 ( Prior studies have implicated TrkB in the survival of brain tumor initiating cells in the absence of growth factors epidermal growth factor (EGF) and fibroblast growth factor (FGF)11, while more recent work has implicated TrkB and its ligand, brain-derived neurotrophic factor (BDNF), in the crosstalk between glioma stem cells and their differentiated glioblastoma cell progeny12, suggesting that this neurotrophin receptor exhibits complex interactions within the brain tumor environment that extend beyond the canonical TrkB-BDNF signaling events characterized in normal neurodevelopment. Malignant tumors of the central nervous system and brain tumors, specifically, result in the highest years of potential life lost compared with other cancer types13, while glioblastoma multiforme (GBM), in particular, remains the most common malignant primary brain tumor with a mere 2–4% 5-year survival rate14. We sought to further understand the complex role of TrkB in GBM and lower grade gliomas (LGGs) in effort to learn more about the neurotrophin receptor splicing contributions to these devastating tumors.As decades of research have uncovered key drivers in oncogenic signaling through the discovery of tumor suppressors, oncogenes, histone modifications, DNA methylation, and environmental factors, the possibility remains that such factors may contribute to splicing events at the core of oncogenesis. While recent work independently highlights the evolving importance of aberrant splicing in cancerNTRK2 gene  has well known roles in neuronal survival, proliferation, differentiation, apoptosis, and exerts diverse effects on cellular and neural outcomes15. In addition to the full-length receptor tyrosine kinase, TrkB.FL, several lesser known, alternatively spliced variants, including the truncated isoform, TrkB.T1, have been shown to exist17. Once thought dominant-negative due the absence of a kinase domain, TrkB.T1 shares the same extracellular and transmembrane domains, as well as the first 12 intracellular amino acids, as other variants yet contains a unique C-terminal sequence of 11 amino acids that is conserved across species from rodents to humans18. In vitro, TrkB.T1 has been shown to alter Ca2+ signaling19, regulate neuronal complexity21, influence astrocytic morphology via Rho GTPases22, modify filopodia outgrowth23, and contribute to signal transduction and proliferation25, raising the possibility that this formerly considered dominant-negative receptor variant has unique and important roles in both normal and abnormal brain development.The neurotrophin receptor TrkB, encoded by the NTRK2 splice variant TrkB.T1 in gliomas and highlight the importance of exploring alternative splicing of TRKs in basic and translational research.Here, we show that the TrkB.T1 splice variant is the predominant TrkB isoform expressed across a range of human gliomas. By generating an antibody specific for this splice variant, we show that TrkB.T1 receptor localization differs between normal, healthy brain regions and gliomas, in both rodents and humans. In vivo experiments using RCAS-tv/a technology demonstrate that TrkB.T1 enhances PDGFB-driven tumors in mice, while in vitro experiments show that TrkB.T1 enhances the perdurance of PI3K and STAT3 signaling pathways including pAkt and pS6rp. Together, these results demonstrate a previously unidentified role for the 26, PCA of gene expression data from GBM and LGG samples also yielded distinct clusters of gliomas based on status of CpG island methylator phenotype (CIMP) and non-CIMP  Project on 1216 normal samples across 13 GTEx-defined brain regions Table . For a mMP Table . To ruleNTRK2 splice variants29, the full-length receptor tyrosine kinase (TrkB.FL), and kinase-deficient truncated isoform (TrkB.T1). Using publicly available transcript data from GTEx (available as RPKM data) and TCGA (available as RSEM counts from the legacy archive), we converted all transcript data to Transcripts Per Million (TPM) to allow for isoform comparisons across TCGA and GTEx30. Contrary to existing hypotheses surrounding the full-length kinase, TrkB.FL, as the sole suspected NTRK2 contribution to oncogenesis, TrKB.FL levels remain relatively consistent across pooled normal supratentorial regions, LGG and GBM   analysis to determine which, if any, classes of genes were enriched in NTRK2 glioma pairings ngs Fig. . GO analngs Fig. , suggest34, little insight could be gained as to which TRK (or TRKs) were present or if receptor distribution differed between neural and non-neural tissue. Further, TrkB-specific antibodies do not easily discriminate between the full and truncated variant gene products as the majority are generated against either the entire extracellular domain or extracellular subdomains—regions conserved between full-length and various truncated isoforms35  was procured and subjected to TrkB.T1 immunohistochemistry (IHC) with this TrkB.T1-specific antibody. Each control case contained both supratentorial and infratentorial brain regions, including cerebral cortex, cerebellum, hippocampus, subiculum, entorhinal cortex, and thalamus ; Astrocytoma/Glioblastoma, IDH-mutant (n = 5); and Astrocytoma/Glioblastoma, IDH-wild-type (WT) 38. Pathological review of tumor sections confirms that TrkB.T1 staining is intensely diffuse throughout all CIMP and non-CIMP tumors. Specifically, these tumors lack punctate intracellular clustering of the receptor seen in non-neoplastic cells, demonstrating that both the expression and distribution of TrkB.T1 differ in normal brain compared with gliomas, providing a degree of visual specificity and receptor localization that was previously not possible. Diffuse gliomas of all classifications demonstrated patchy to widespread, moderately intense, cytoplasmic TrkB.T1 immunohistochemical staining with noticeable lack of normative vesicular pattern 1 N/tv-a (Nestin-expressing cell of origin) wild-type mice significantly enhanced glioma formation as evidenced by decreased survival in mice injected with RCAS-PDGFB + RCAS-TrkB.T1 compared with mice injected with RCAS-PDGFB only   Fig. . In addi05) Fig. , additio05) Fig. , b suggeNTRK2 transcript-level analysis of normal brain and human gliomas reveals relatively low amounts of full-length TrkB.FL compared with abundant amounts of TrkB.T1 in tumor tissue, suggesting that this isoform has an important role in both normal neurobiology and oncogenesis alike. Subcellular distribution patterns of this TrkB.T1 suggest potentially distinct roles for neuronal and glial cell populations in normal brain, whereas as the lack of vesicular pattern in CIMP and non-CIMP tumors highlights the need for further characterization of differences in signaling mechanism as a result of receptor compartmentalization. As mentioned above, in addition to endocytic and vesicular compartment GO terms . In order to explore if TrkB.T1 can enhance growth factor signaling in vitro, 3T3/tv-a cells were treated with PDGF-BB ligand, chosen for its role in glioma biology, for varying durations and lysates were subjected to western blot analysis to explore downstream targets often implicated in gliomagenesis. TrkB.T1-expressing 3T3/tv-a cells showed significantly enhanced and sustained levels of phospho-STAT3, phospho-AKT, phospho-S6rp, and slight increases in phospho-ERK in response to PDGF treatment 47. Western blot analysis of phosphorylated (p)PDGFR-β and total PDGFR-β demonstrate that there is enhanced pPDGFR-β in TrkB.T1-expressing 3T3/tv-a cells and also a maintained expression of total PDGFR-β upon ligand stimulation  < 0.05 with a fold change of more than 2 in each respective direction)  < 0.05 with a fold change of more than 2 in each respective direction) (See Supplementary Data To explore the correlations between TrkB.T1 levels and GO terms in human tumor initiating GBM stem cells (GSCs), we took whole gene expression data from four previously publishednes Fig.    Fig. . GO enri5%) Fig. , suggestTreatment of NSCs transduced with TrkB.FL and TrkB.T1 showed that both lines were sensitive to phosphoinositide-3 kinase (PI3K) and mTOR inhibitors  in a dose dependent manner and that TrkB.T1 NSCs were marginally, but statistically significantly, more sensitive to LY294002 than TrkB.FL NSCs at both 48 and 72 h . In addition to altered receptor localization, TrkB.T1 is not only elevated in human gliomas, but also in murine gliomas driven by multiple genetic mechanisms, and across human and rodent GSC and tumorsphere lines . CIMP and nonCIMP status for TCGA-GBM58 and TCGA-LGG59 samples were obtained from Bolouri et al.26 Whole gene RPKM read counts for The GTEx Project (v6)60 was obtained from https://gtexportal.org/ (version 6) on 03/30/2017 (dbGaP accession number phs000424.v7.p2). GTEx is supported by the Common Fund of the Office of the Director of the National Institutes of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. Whole gene counts from GTEX and TCGA was transformed to log2(rpkm + 1) using R .Obtaining and transforming transcript level data. Transcript Data (TCGA RNAseqV2 RSEM data) for TCGA-GBM and TCGA-LGG was downloaded from the legacy archive of TCGA. Transcript data (RPKM data) was also downloaded from GTEx(v6) website. To make the RSEM data from TCGA, and RPKM data comparable, we converted both data sets to TPM (transcripts per million). The RSEM counts from TCGA to TPM counts using the following formula30: RSEM can be multiplied by 106. The RPKM transcript data from GTEX was converted to TPM data using the formula30: TPM = RPKM/ × 106. For transcript analyses, NTRK2 transcript IDs were manually aligned to confirm sequence homology and are as follows: TrkB.FL , TrkB.T1 .RNA Seq analysis for 448 vs 559 and G14 vs G179 GSC cell lines. Raw gene counts for cell line 448, 559, G14, and G179 were obtained from GEO repository, (GSE89623)61.Cufflinks62 results for TrkB.T1 were used to differentiate the 4 cell lines into TrkB.T1 High  and TrkB.T1 Low  groups. Differential Gene Expression analysis was performed between TrkB.T1 High and TrkB.T1 Low  cell lines using the R/Bioconductor package DESeq263 and a statistical cutoff of FDR < 0.05 and FC > 2 was applied to obtain differentially regulated genes.RNA Seq analysis for NSC cell lines. RNA-seq reads were aligned to the UCSC hg19 assembly using STAR264 and counted for gene associations against the UCSC genes database with HTSeq65. Differential Gene Expression analysis was performed between GFP vs TrkB.T1 NSCs, GFP vs TrkB.FL NSCs, and TrkB.T1 vs TrkB.FL NSCs, using the R/Bioconductor package edgeR66 and a statistical cutoff of FDR < 0.05 and FC > 1.25 was applied to obtain differentially regulated genes. Raw data files have been deposited to the NCBI Gene Expression Omnibus under accession number GEO: (GSE136868)Downstream analysis. For PCA plots, the R function ‘dist’ was used to calculate Euclidean distance between samples. We computed the PCA for the above distances and visualized it using R package ggplot2 (v 2.2.1). Transcript Data was visualized using boxplots using R package ggplot267. Whole gene correlations between Normal Brain Data from GTEX and LGG, GBM data from TCGA was calculated using R package DGCA (https://cran.r-project.org/web/packages/DGCA/index.html). For predominant transcript analysis, log2 TPM values for each transcript were compared and each sample from pooled normal brain (GTeX), LGG (TCGA) and GBM (TCGA) was marked as either TrkB.T1 or TrkB.FL, depending on which transcript, of the two, had the greater value. GO and Reactome Pathway enrichment analysis69 was done using R Bioconductor Packages clusterProfiler v 3.4.470 and dot plots were made using R Bioconductor package DOSE71. Heatmaps were made using R package pheatmap (https://CRAN.R-project.org/package=pheatmap).GSC transcript analysis. The human GSCs dataset in Fig. 31 and 6 additional lines  from GSE150653. For the newly generated data, sequencing libraries were prepared with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760) as recommended by the kit manufacturer and then sequenced with a Illumina NexSeq 550. RNA-seq samples were analyzed using the nextpresso pipeline72 as follow: reads were aligned to the human genome (hg19) with TopHat-2.0.1073 using Bowtie 1.0.074 and SAMtools 0.1.1975, allowing 3 mismatches and 20 multi-hits; transcripts quantification was calculated with Cufflinks 2.2.173, using the human hg19 transcript annotations from https://ccb.jhu.edu/software/tophat/igenomes.shtml. Transcriptional subtypes were obtained using the ‘ssgsea.GBM.classification’ R package76, through the SubtypeME tool of the GlioVis web portal (http://gliovis.bioinfo.cnio.es)77.56, deposited in GSE110700, were analyzed for NTRK transcript expression, as above, and subjected to immunoblotting and RT-qPCR.Mouse tumorspheres from ref. Immunoblotting. Lysates were prepared in RIPA lysis buffer  and protein concentrations were determined by DC protein assay kit . Proteins were run on a 8% SDS-PAGE gel and transferred to nitrocellulose membrane . After blocking the membrane in 5% milk, 0.1% Tween, 10 mM Tris at pH 7.6, 100 mM NaCl, primary antibodies TrkB  and vinculin  were added. Anti-mouse or rabbit-HRP conjugated antibodies (Jackson Immunoresearch) were used to detect desired protein by chemiluminescence with ECL .Reverse transcription quantitative PCR RNA was isolated with TRIzol reagent  according to the manufacturer’s instructions. For reverse transcription PCR (RT-PCR), 1 μg of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit . Quantitative PCR was performed using the SYBR-Select Master Mix  according to the manufacturer’s instructions. qPCR was run using specific primers for TrkB.T1 (qmNtrk2T1Fw_CAGGTAGAACGGAGCAGCA and qmNtrk2T1RevA_GGTTAGCAGAGGGCAATGGA) and the TrkB.FL (qmNtrk2FLFwdA_TTCCTTGCCGAGTGCTACAA and qmNtrk2FLRevA_TCGTGCTGGAGGTTGGTC). The threshold cycle number for the genes analyzed was normalized to GAPDH (mGAPDHFwd_TCAACAGCAACTCCCACTCTTCCA and mGAPDHRev_ ACCCTGTTGCTGTAGCCGTATTCA).36  and Lino Tessarollo, National Cancer Institute ) were immunized with TrkB.T1 peptide Ac-C(dPEG4)FVLFHKIPLDG-OH maleimide coupled to KLH carrier protein. Following a 12+ week boosting protocol splenocytes were isolated from high titer mice and electrofused to a subclone of the P3x63-Ag8 myeloma cell line . Hybridomas secreting peptide specific antibody were identified and isolated using a ClonePix2  colony picker. Antibodies from the picked clones were validated for peptide binding by flow cytometry using a cytometric bead array carrying the target peptide. Clone FH1D12 was subcloned (CPII) followed by validation for peptide binding by bead-based flow cytometry. Affinity purified IgG2b lambda from the hybridoma was further characterized by Western blot analysis and IHC tissue staining. Antibody development was performed in Holland Lab and with the Antibody Technology Resource (Fred Hutchinson Cancer Research Center).Murine monoclonal antibody (clone FH1D12) was generated at the Fred Hutchinson Antibody Technology Core Facility. Briefly, male and female 20-week-old C57BL6 mice that were characterized as genetic null for TrkB.T1Wes™ capillary electrophoresis for antibody development. Protein Simple Wes™ was used at two stages of antibody screening: Stage 1, selecting immunized mice to advance for hybridoma generation, and Stage 2, for validation of monoclonal antibodies that Western blot full-length TrkB.T1 protein in murine brain detergent extracts. Briefly, mouse brain lysates from TrkB.T1+/+ and TrkB.T1−/− mice were prepared by mechanical disruption  in RIPA lysis buffer  containing HALTTM Protease and Phosphatase Inhibitor Cocktail and EDTA (ThermoScientific™) and normalized to 2 μg/μl total protein via Bradford Protein Assay. Approximately 8–10 ng total brain extract protein was loaded into the Protein Simple Capillary WES system for Western analysis following Protein Simple recommended methods78. Mice with antisera (1:2500 dilution in PBS and Antibody Diluent (Protein Simple)) that identified a positive band at 90–100 kDa for the TrkB.T1 protein were advanced forward for final boosting, splenectomy, and hybridoma generation  antibody were sequenced using rapid amplification of cDNA ends (RACE) cloning with standard primer sets and the SMARTer™ RACE 5′/3′ kit (Clonetech). The variable regions were then formatted into a fusion construct containing a rabbit antibody Fc domain 79. Following purification, the protein was supplemented with 5% glycerol, snap frozen using liquid nitrogen, and stored at −80 °C. Final stocks were 11.5 mg/ml in 85 mM Sodium Citrate 140 mM Tris-HCl with 5% glycerol.Antibody validation phase – large batch IHC. Four-µm sections of a multi-tissue block containing human brain, mouse brain, TrkB.T1−/− mouse brain, a cell pellet over-expressing TrkB.T1 and a cell pellet without TrkB.T1 were cut and stained with the Leica Bond Rx . Slides were pretreated with H2 antigen retrieval buffer for 20 min. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 5 min. A TCT protein block was applied for 10 min . Supernatants from multiple boosting and pre-/post-fusion samples, along with antibody clone D12 were used at 1:20 and applied to the tissue for 30 min. The antibody was then detected using Leica Power Vision HRP Mouse specific polymer  for 12 min and staining was visualized with Refine DAB  and a hematoxylin counterstain was used . Mouse isotype control slides were included for each run (Jackson ImmunoResearch Laboratories) at 2 μg/ml. Digital images of IHC-stained slides were obtained at ×40 magnification (0.25 µm/pixel) using a whole slide scanner  fitted with a 20×/0.75 Plan Apo objective lens and ×2 magnification changer . Images were saved in SVS format (Aperio), managed with server software , and retrieved with a file management web interface . Histology and IHC for antibody validation was performed by Experimental Histopathology Shared Resources at Fred Hutchinson Cancer Research Center.Mouse brain, mouse glioma, human brain, and human glioma. Immunohistochemical staining was performed on 5um formalin-fixed/paraffin-embedded tissue sections using a Discovery XT Ventana Automated Stainer ., run using standard Ventana reagents and the Discovery ChromoDAB and Discovery OmniMAP anti-ms HRP kits to ameliorate non-specific mouse-on-mouse background and standard Ventana reagents and Vector secondaries for staining with the TrkB.T1 SPEH1_D12 scFv-Fc fusion.n = 5); Astrocytoma, IDH-mutant (n = 5); and Glioblastoma, IDH-WT 80. Normal brain control tissue from rapid autopsies (n = 5) was procured from the University of Washington’s Neuropathology Core Brain Aging and Neurodegeneration Brain Bank. Each control case contained multiple brain regions, including cerebral cortex, cerebellum, hippocampus, subiculum, entorhinal cortex, and thalamus.Approval for the use of human subject material was granted by the University of Washington’s Institutional Review Board (Study #44806 and #00002162). Archived FFPE pathology tissue were reviewed by a board-certified neuropathologist (P.J.C.) and classified according to the 2016 WHO classification of CNS tumors: Oligodendroglioma, IDH-mutant and 1p/19q-codeleted  were removed, fixed in 10% neutral-buffered formalin for a minimum of 72 h and embedded into paraffin blocks. 5 µm serial sections were cut from formalin-fixed paraffin-embedded specimens and mounted on slides.Cell culture. GSC and NSC isolates were grown in NeuroCult NS-A basal medium (StemCell Technologies) supplemented with B27 (ThermoFisher), N2 (2× stock in Advanced DMEM/F-12 (ThermoFisher) containing 25 μg/mL insulin, 100 μg/mL apo-transferrin, 6 ng/mL progesterone, 16 μg/mL putrescine, 30 nM sodium selenite, and 50 μg/mL bovine serum albumin (Sigma)), and EGF and FGF-2 (20 ng/ml) (PeproTech) on laminin (Trevigen or in-house-purified)-coated polystyrene plates and passaged81. Cells were detached from their plates using Accutase (EMD Millipore).Lentiviral production and infection. For virus production, pLJM1 (Addgene) constructs containing the NTRK2 inserts of interest  were transfected into 293T cells, along with psPAX and pMD2.G packaging plasmids (Addgene), using polyethylenimine (Polysciences). Fresh media was added 24 h later and viral supernatant harvested 24 h after that. For infection of NSC-U5 cells, 1e5 cells/well were seeded into 6-well plates. Lentivirus was used unconcentrated and cells were infected at a MOI < 1 24 h after seeding. 72 h after seeding, selection was begun for cells successfully expressing the constructs using 2 μg/mL puromycin (for 3 days). Cells were expanded after selection to create a stable line and collected for RNAseq three weeks post infection.PI3K and mTOR inhibitors. In order to assess the sensitivity of TrkB.T1 and TrkB.FL overexpressing NSCs to LY294002 and rapamycin, we used biological triplicates of each line and seeded 6 × 4000 cells per triplicate into four laminin coated 96-well plates. One day after seeding, LY294002  and rapamycin  were dissolved in DMSO warmed to 37 degrees at 10 mM, diluted to 10 μM in NSC media and added to cells in 100 μl NSC media for a total volume of 200 μl at specified concentrations. After 48 and 72 h of drug treatment, plates were analyzed using CellTiter-Glo® Luminescent Cell viability Assay . In brief: CTG reagent was reconstituted according to the manufacturer’s instructions. The reagent was then diluted 1:5 in PBS. The 96-well plates were equilibrated to room temperature and existing NSC media with drugs was removed before 100 µl of diluted CTG reagent was added to each well. The cells in the plates were then lysed for 2 min with shaking, followed by a 10 min incubation at room temperature. 90 µl of the cell lysate in each well was transferred to opaque 96-well plates for detection. The emitted ATP-driven luminescence signal was detected using the Synergy 2 instrument (BioTek) and the Software Gen5. Integration time was set at 1 s and sensitivity to 135.23), visualized on a 1% agarose gel and extracted via QIAquick gel extraction kit (Qiagen), and ligated into modified RCAS (replication-competent avian sarcoma-leukosis virus (ASLV) long-terminal repeat with splice acceptor) vectors82. Appropriate inserts were confirmed by sequencing at Fred Hutch Genomics Core. RCAS virus was produced in DF-1 packaging cells with minor modification84. In brief, DF-1s (ATCC) were maintained in Dulbecco’s Modified Eagle Media (DMEM) with 1% penicillin/streptomycin and 10% fetal bovine serum (Paa Laboratories) at 39 °C, were transfected with RCAS-TrkB.T1 using X-tremeGENE9 (Roche) + Opti-MEM and passaged three times at high confluency to enhance viral propagation. Expression for TrkB.T1 was confirmed by western blot after three passages while TrkB.FL was at maximum size limitations for RCAS and did not make TrkB.T1 protein.Flag-tagged TrkB.T1 and Flag-tagged TrkB.FL DNA were cut from pEFBOS vectors  using Extreme-Gene 9Transfection reagent (Roche) accordingly to manufacturer’s protocol. The cells were maintained for three passages to ensure viral propagation to all cells. After confirmation of RCAS-inserts by western blot, DF1s (passage 4 or later) were used for injection into murine brain. Newborn Nestin(N)/tv-a(agouti) pups  were injected intracranially (Hamilton syringe #84877) with 1 μL of approximately 1 × 105 DF-1 cells infected with and producing relevant RCAS viruses suspended in serum-free DMEM (RCAS-TrkB.T1 and RCAS-PDGFB). Simultaneous delivery of two RCAS viruses was performed by the injection of 1 μL of ~2 × 105 DF-1 cells mixed with equal ratio. Mice were monitored for weeks or months to check for tumor related symptoms such as palpable masses, lethargy, weight loss, seizure, hyperactivity, altered gait, poor grooming, macrocephaly, paralysis. Mice with severe hydrocephalus presumably due to injection trauma or an inflammatory response against the DF-1 cells were excluded from survival analysis in this study. All animal experiments were approved by and conducted in accordance with the Institutional Animal Care and Use Committee of Fred Hutchinson Cancer Research Center (protocol #50842).We utilized the RCAS/tv-a system for murine tumor modeling in immunocompetent miceTv-a receptor39 were subjected to retroviral infection with RCAS-TrkB.T1 via 3 × 8 h cycles using sterile filtered, viral-containing supernatant from DF-1 cells supplemented with 8 μg/ml polybrene (Sigma) and later selected with 2.5 μg/ml puromycin (GIBCO). 3T3/tv-a cells were maintained for three passages prior to any experiments and cultured in DMEM containing penicillin/streptomycin and 10% fetal calf serum . Prior to PDGF treatment, 3T3-tva cells were serum-starved for 24 h. Recombinant human PDGF-BB  was diluted in fresh serum-free DMEM and added for various time points before cells were lysed on ice in RIPA extraction buffer  after being rinsed twice with Dulbecco’s phosphate buffered saline (DPBS). Extracts were further lysed by mechanical disruption using 18 ½ gauge needles, rotated at 4 °C for 20 min to ensure lysis, and clarified by centrifugation . Proteins were resolved by SDS/PAGE  according to XCell Sure Lock™ Mini-Cell guidelines, blocked with 5% milk/TBST and probed with specified antibodies overnight at 4 °C in 5% BSA/TBST. After three TBST rinses, species-specific secondary antibodies were added in 5% milk/TBST at 1:10,000. Blots were rinsed three times with TBST before being developed with Amersham™ ECL™ Western Blotting Detection Reagents . For densitometric analyses, immunoreactive bands were scanned and quantitated using National Institutes of Health ImageJ (Scion).Stable NIH-3T3 cells expressing ® #4370; lot: 12 at 1:2000), β-actin , pS6 ribosomal protein , TrkB , TrkB , TrkB , pPDGFR β , PDGFR β , ERBB3 , NRG2 , vinculin  and added in 5% BSA/TBST overnight at 4 °C.For TrkB.T1 IHC, the generated mouse TrkB.T1 antibody was used (1.3 mg/ml) at 1:20 and recombinant rabbit fusion SPEH1_D12 (11.5 mg/ml) was used at 1:500 and TrkB kinase antibody  at 1:250. For western blot analyses of human brain, mouse brain, and 3T3-Tv-a cells, commercial antibodies were used according to manufacturer specifications and bands were confirmed by size using Spectra™ Multicolor Broad Range Protein Ladder : pSTAT3 , pAkt , pERK  (Thr202/Tyr204) (D13.14.4E) XPNestin(N)/tv-apups at P0. A minimum of four P0 N/tv-a WT pups were used. SVZ/Hippocampal-centric region was dissected and placed immediately in 3 ml of accutase warmed to 37 °C. Cells were pipetted 30 times for mechanical dissociations and left in a 37 °C water bath with occasional agitation before being pipetted an additional 30 times with a P1000 to create a single cell suspension. Two additional mLs of NSC media were added, cells centrifuged @ 200 × g for 3 min at 4 °C. Supernatant was removed and pellet was resuspended in 5 ml NSC media, transferred to a new tube (leaving any debris behind) and seeded 100 μL into 4 ml NSC media in 25 cm2 flask. 50 ml fresh NSC media were made each day: 50 ml NSC media consisted of , 50 μL EGF (20 μg/ml stock), 50 μL bFGF (10 μg/ml stock), 125 μL Heparin (Stem Cell Technologies #07980), 500 μL P/S). Fresh growth factors were added every three days. Neural progenitors were passaged twice prior to the start of experiments. For retroviral infection of murine neurospheres with RCAS-TrkB.T1 or RCAS-GFP, RCAS virus was produced in DF-1 packaging cells maintained with serum-free neurosphere medium and was then diluted 1:1 with collection media (fresh murine neurosphere media). Efficiency of retroviral infection was confirmed via western blot after one passage and images were obtained in Fred Hutch Imaging Core.Neural progenitors were isolated from Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


Necrobiosis lipoidica‐like lesions, in known cases of sarcoidosis, can be considered as a member of the broad spectrum of histologic changes in sarcoidosis. Necrobiosis lipoidica‐like lesions, in known cases of sarcoidosis, can be considered as a member of the broad spectrum of histologic changes in sarcoidosis. The patient was a known case of systemic sarcoidosis for 10 years. She was referred to our clinic with a 2‐month history of two yellow‐red plaques on her right lower limb, exactly located on the anterior part of the right thigh and right knee  all were in the normal range. She did not also mention any personal or family history of diabetes mellitus. Other laboratory data including serum calcium level, 25‐hydroxyvitamin D level, ESR were in the normal range. However, angiotensin‐converting enzyme (ACE) was above the normal range. Her clinical eye examination revealed no abnormality related to sarcoidosis, but her chest X‐ray demonstrated bilateral hilar lymphadenopathy.The systemic treatment of sarcoidosis continued as before, with no alterations. Also, the topical steroid was prescribed for her skin lesions and no marked improvement was detected after several months.3Necrobiosis lipoidica diabeticorum was first described in 1929 as a cutaneous granulomatous lesion, sometimes associated with diabetes mellitus.Herein, we report a rare case of systemic sarcoidosis that developed necrobiosis lipoidica‐like lesions in a nondiabetic patient. There are limited reports of NL development with concurrent sarcoidosis. We discuss 14 reported cases of this association in the literature Necrobiosis lipoidica usually presents as yellow‐brown to violaceous papules that develop into atrophic plaques with telangiectasia. Cutaneous sarcoidosis presents as red‐brown to violaceous papules or plaques, superficial nodules, or erythema nodusom. Various cutaneous manifestation of sarcoidosis is the possible explanation for describing it as the “great imitator” including even the atrophic telangiectatic plaques of NL, making its clinical diagnosis more difficult.Moreover, in microscopic examinations, collagen necrobiosis in lower dermis, foamy histiocytes, and lymphoplasmacytic infiltrations, all organized in horizontal layers, is characteristic of NL lesions while the presence of epithelioid‐cell granulomas is characteristic for sarcoidosis lesions.Thus far, different theories have been suggested to explain the coexistence of multiple cutaneous granulomatous diseases. In previous reports, sarcoidosis has been associated with other granulomatous diseases such as granuloma annulare, rheumatoid arthritis, and Crohn's disease. The possible explanation for these associations may be attributable to the similar cellular signaling mechanisms of granulomatous formation. In this regard, Macaron et al,Moreover, Ball et alNone to declare.FA: involved in data gathering and preparing the manuscript. MT: involved in data gathering and preparing the manuscript.AR: involved in histopathology evaluation and preparing the manuscript. SD: involved in preparing the manuscript. RMR: involved in data gathering, supervising the whole report, and preparing the manuscript.The ethical issues were completely considered to prepare this case report according to our institution ethical board guidelines. Moreover, this article was prepared regarding the declaration of Helsinki.