PD-L1 is a well-known immune co-stimulatory molecule that regulates tumour cell escape from immunity by suppressing the immune response. However, the clinical significance of PD-L1 in the progression of ovarian cancer is unclear. Our study demonstrated that PD-L1 is up-regulated in ovarian tumour tissue compared with its expression level in adjacent normal tissue. Furthermore, we confirmed that PD-L1 increases the proliferation of cancer cells by activating the AKT-mTORC signalling pathway, which is also enhanced by the expression of S6K, the substrate of mTORC. In addition, PD-L1 promotes the autophagy of ovarian cancer cells by up-regulating the expression of BECN1, a crucial molecule involved in the regulation of autophagy. In conclusion, PD-L1 may provide a target for the development of a novel strategy for the treatment of ovarian cancer. Ovarian cancer (OC) is one of the most common malignant tumours in women, and its mortality rate ranks first among gynaecological tumours ,2. Many The PI3K–AKT signalling pathway is crucial for regulating many cellular functions, which it does by modulating the phosphorylation, transcription and translation of the downstream targets necessary for catabolic and anabolic processes . PreclinRecent studies have shown that immunosuppression provides an opportunity for cancer to progress , and somOvarian cancer samples were obtained from Shaanxi Nuclear Industry 215 Hospital. Written informed consent was obtained from the patients, and the study was approved by the Institutional Human Experiment and Ethics Committee of Shaanxi Nuclear Industry 215 Hospital. The Institutional Review Board (IRB) approval number is 2017SNIH-034.The ES2 cell line was purchased from ATCC (CRL-11731) and maintained in DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic solution (100 U/ml penicillin and 100 μg/ml streptomycin). Transfection was performed with Lipofectamine TM 2000  according to the manufacturer’s instructions.IFN-γ (Cat. # IFG-H4211) was purchased from ACROBiosystems . MK-2206 (Cat. #HY-10358) and WYE-687 (Cat.# HY-15271) were purchased from MCEy .Cells were seeded at a density of 3000 cells per well in 96-well culture plates and treated with a series of IFN-γ concentration levels for a specific duration or administered at different time points. After treatment, the cells were incubated with fresh media containing CCK-8 at 37°C for 3 h. Then, the absorbance was measured at 570 nm and the results were recorded by an enzyme-linked immunosorbent assay reader.To assess the level of cell proliferation, ES2 cells were seeded in 96-well plates. The cells were incubated in complete media under standard conditions. After transfection, the cell assays were performed on specific days, as mentioned above. Forty-eight hours after transfection, cell proliferation was measured by incorporating 5-ethynyl-2′-deoxyuridine (EdU) with an EdU cell proliferation assay kit . Briefly, the cells were incubated with 40 µM EdU for 5 h and then fixed, permeated and stained with EdU according to the manufacturer’s protocol. The nuclei were stained with DAPI (Sigma) at a concentration of 1 µg/ml for 30 min. The proportion of the cells incorporated with EdU was measured with fluorescence microscopy.3VO4, and 1 mM leupeptin cocktail]. Thirty micrograms of protein were loaded onto 10% polyacrylamide gels for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gel was transferred onto a nitrocellulose membrane, which was blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C.The protein was extracted with lysis buffer [20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, NaAfter washing with TBST, the membrane was incubated with a secondary antibody for 1 h at room temperature. The signalling was detected with an enhanced chemiluminescence HRP substrate by Western blotting . The following primary and second antibodies were purchased from Proteintech : PD-L1 , S6K , BECN1 , p-AKT , AKT , LC3 , GAPDH , HRP-linked anti-rabbit secondary antibody  and HRP-linked anti-mouse secondary antibody . P-S6K  were purchased from Cell Signaling Technology, Inc .Cq method was used to process the data and calculate the relative gene expression. The following human primer sequences were used: PD-L1 forward: 5′-CAAATGTTGTTTGGGTCATGC-3′ and reverse: 5′-GTAAAACGACGGCCAGTCATTCCTTCCTCTTGTCACGC-3′ and GAPDH forward: 5′-GGCATGGACTGTGGTCATGAG-3′ and reverse: 5′-TGCACCACCAACTGTTAGC-3′.Total RNA was extracted from human ovarian tumour tissues using TRIzol reagent . Reverse transcription was performed by using HiScript Reverse Transcriptase following the manufacturer’s instructions . Three micrograms of total RNA was used for the reverse transcription reaction. Quantitative PCR was performed using SYBR Green Real-Time PCR Master Mix . All values of sample expression were normalized to that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) . The following PCR protocol was used: 5 min at 95°C, followed by 32 cycles at 95°C for 15 s, 56°C for 15 s and 72°C for 1 min. The ∆∆The core tissue biopsy sample exhibiting carcinoma with a 2.5-mm diameter was punched from individual donor paraffin-embedded tissue blocks and arranged accurately into a new recipient block. A sample of 4 μ was cut and used for IHC analysis. PD-L1 expression was detected via immunohistochemical staining using streptavidin peroxidase. Briefly, the slices were de-waxed with xylene and rehydrated through an ethanol gradient wash. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 min. After antigen retrieval in 10 mmol/l citrate phosphate buffer for 5 min, the sections were blocked in 10% normal goat serum for 1 h. The sections were then incubated with PD-L1 antibody at 4°C overnight. Vectastain® Elite ABC kits (Peroxidase)  were used for the detection of PD-L1 signalling.t test or one-way ANOVA was used to analyse quantitative variables. The Kaplan–Meier method was used to evaluate survival curves, and the log-rank test was used to test the differences between the survival curves. A result was considered statistically significant when a bilateral P-value was less than 0.05.All results were confirmed through replication of at least three independent experiments, and all quantitative data are presented as the mean ± SD. Student’s To investigate the influence of PD-L1 on human ovarian cancer progression, IHC was performed to examine the protein expression level of PD-L1 in five cases of paraffin-embedded ovarian tissues. PD-L1 expression was significantly increased in primary tumour tissues compared with adjacent normal tissues (ANT) A, supporTo further explore the biological role of PD-L1 on the progression of human ovarian carcinoma, we used a cell model in which the up-regulated expression of PD-L1 was stimulated by IFN-γ . In thesPrevious studies have reported that the mTORC1 pathway plays an important role in regulating cell autophagy and proliferation ,34,35; wOveractivation of the PI3K–AKT cascade is one of the most common events in human cancer progression . To furtPrevious experiments demonstrated that PD-L1 not only affects autophagy in ovarian carcinoma by activating the AKT–mTORC pathway, but also increases cell proliferation by inducing the phosphorylation of S6K. Therefore, we hypothesized that S6K, the kinase that regulates proliferation and protein synthesis, has an impact on PD-L1 expression, and the results from Western blotting proved that the inhibition of mTORC significantly reduced the protein levels of PD-L1 A. FurtheOC is one of the most common malignant tumours in women and ranks first in mortality among gynaecological tumours ,42. TherCollectively with the other report, the ribosomal protein S6K (S6 kinase) represents an extensively studied effector of the target of rapamycin complex 1 (TORC1), which plays important roles in cellular and organismal physiology ,47. The BECN1 is a well-established regulator of autophagy, and Beclin-1 could be affected by a number of molecules in the cell, which can lead to either activation or inhibition of autophagy . TRIM59 In summary, the present study identifies the regulatory link between PD-L1 and mTORC/S6K and describes a potential mechanism explaining PD-L1, which contributes to ovarian cancer growth and cell proliferation. Our study demonstrates that the expression of PD-L1 is significantly up-regulated in clinical OC tissues and is positively associated with the phosphorylation of S6K1. Furthermore, PD-L1 and S6K constitute a pathway that affects the proliferation of cancer cells. Therefore, PD-L1 has important value as an indicator of progression in OC patients and may become a therapeutic target in the future.


OBJECTIVES/SPECIFIC AIMS: The objectives for the Rigor, Reproducibility, and Transparency course within KAIZEN-Edu was to provide a platform that allows essential training, in a novel and customizable approach, for a large number of students across the multiple institutions within the UAB CCTS Partner Network. Successful implementation across this geographically diverse of partner institutions would serve as proof of concept to future dissemination across the CTSA consortium. METHODS/STUDY POPULATION: We used the “build a game” tools within Kaizen-Edu to design the “Rigor and Reproducibility Game.” The games consisted of four modules, with 20 questions designed to test participant knowledge, and edify learners on particular concepts through a multimedia approach  with content provided as questions released over 4 weeks. Researchers from across the UAB CCTS Partner Network developed comprehensive modules for (1) How Scientists Fool Themselves/Scientific Premise, (2) Authentication of Chemical and Biologic Resources and Sex and Other Biologic Variables, (3) Statistical Rigor, and (4) Comprehensive Review. A typical week began with review articles (1–2) sent to each participant. The participants are informed that 5 questions will be released midweek testing the key concepts from the papers. When ready, the participant logs into Kaizen-Edu and starts to answer questions/play the game. Immediately, the articles are opened for reference, followed by a brief 4–5 minute video which reinforces key concepts and then timed questions begin. A typical question is allowed 3 minutes (visible countdown clock). Accurate responses result in the addition of points, with double points awarded for correct answers within the questions time limit. No points are awarded for incorrect answers. After each question, a detailed explanation reviews and reinforces the key concepts. Each participants’ points contribute to both their individual score and team scores, which influences their position on the Rigor and Reproducibility game leaderboard. RESULTS/ANTICIPATED RESULTS: Within 2017, the Rigor Reproducibility, and Transparency course was conducted 5 times. A total of 126 researchers across 9 institutions were enrolled. A total of 87 enrollees completed the full course, with 80% passing (answering ≥75% of questions correctly) on their first attempt and an additional 20% passing on a second attempt. The distribution of completers across the CCTS Network was UAB=48, Auburn=13, Pennington=10, University of Alabama=5, Hudson Alpha=5, Tulane=4, University of South Alabama=1, LSU=2, and Southern Research=1. Researchers throughout at Partner Institutions represent 46% of the total population trained. DISCUSSION/SIGNIFICANCE OF IMPACT: This software based, gamification-enhanced course was broadly accepted with each session fully enrolled, and learners spread almost evenly between our institution and various Partner Network sites. Our pilot proved that gamification was an effective technique to engage users and produced a high pass rate, suggesting that the content both engaged learners and was effectively internalized. Educational interventions, imbued with principles of gamification provide educators powerful tools that use competition and/or collaboration to disseminate knowledge, engage learners with content, and save educator time as created game content can be reused in additional educational sessions. Analyses of the data trail provided by users engaging with such electronic learning tools will provide educators will insights on how to maximize learning, opening the door to an era of educational analytics.


The monitoring of structures is one of the most difficult challenges of engineering in the 21st century. As a result of changes in conditions of use, as well as design errors, many building structures require strengthening. This article presents research on the development of an externally strengthening carbon-fiber textile with a self-sensing option, which is an idea is based on the pattern of resistive strain gauges, where thread is presented in the form of zig-zagging parallel lines. The first laboratory tests showed the system’s high efficiency in the measurement of strains, but also revealed its sensitivity to environmental conditions. This article also presents studies on the influence of temperature and humidity on the measurement, and to separate the two effects, resistance changes were tested on unloaded concrete and wooden samples. The models were then placed in a climatic chamber, and the daily cycle of temperature and humidity changes was simulated. The research results confirmed preliminary observations of resistivity growths along with temperature. This effect is more visible on concrete samples, presumably due to its greater amount of natural humidity. The strain measurement with carbon fibers is very sensitive to temperature changes, and applications of this method in practice require compensation. About 50 years ago, Concor and Owston  noted thMeasurements performed using resistive strain gauges allow for investigation of changes in strain expressed by the change in resistance. Unfortunately, the measurement of the actual mechanical deformation overlaps with various effects which disturb the measurement signal. These effects mainly consist of environmental influences, such as thermal elongation of the measured object, temperature-dependent changes in strain gauge resistance, thermal shrinkage of the strain gauge measuring film, the thermal response of connecting wires, and surface conductivity with increasing humidity. Carbon-based materials are much more temperature-sensitive than conventional strain sensors made of constantan  . The aboTypically, the effects of temperature and humidity is compensated for in the measurements by combining several strain gauges to form a half or full bridge. Through the skillful combination of strain gauges of the most commonly used Wheatstone bridge circuit , where sBecause identical, or even similar working conditions of measuring and compensating strain gauges may not always be possible, modern computational compensation systems which have been adapted to the thermal and moisture responses of strain gauges are increasingly being used today. In these types of measurements, a quarter-bridge circuit is sufficient, but knowledge of the temperate and humidity response functions is necessary.This paper describes preliminary studies on the temperature-resistant dependence of externally strengthening carbon-fiber textile sensors which have been developed. These tests may be used in the future to build temperature-change compensation functions necessary for use in regard to future devices in engineering practice. They may also be used for development by the authors’ strengthening system, but it is believed that such self-monitoring solutions with reliable information about its deformation in various environmental conditions may be used for future smart structures made out of conductive fibers. Controlling the deformation of FRP (fiber reinforced polymer) composite fibers is much more important than the steels themselves traditionally used in constructions. This is due to their linear elasticity until destruction, which is usually sudden. For this reason, it is advisable to control the strains of the composite fibers to indicate excessive elongation, which may threaten their breaking or delamination.As will be described in this paper, the system takes advantage of two features of carbon fibers, which are their superb strength and electrical conductivity. The idea of measuring strains by recording changes in the resistance is based on operation principles of strain gauges invented in 1938 by Edward E. Simmons and Arthur C. Ruge . Such stIn this way, the first prototype was tested on small models of concrete slabs. To verify the accuracy of the measurement, the device was accompanied by paper strain gauges adhered along its length . A more The production method described was based on traditional plain weaving, where alternating carbon and acrylic threads were arranged as a warp and stabilized by the weft made of cotton. However, this technology has several significant disadvantages. The carbon fiber must be continuous and well-stressed during weaving, which may hinder the automation of production, and only the classic weaving loom can be used. The fiber types used also makes the textile sensor soft, and although this facilitates its application, after adhering, the carbon fibers may not be sufficiently stressed, which can worsen the strengthening efficiency and change the gauge factor. Specifying the gauge factor value is also one of the biggest drawbacks of the developed solution, which is why paper strain gauges were used for the trial tests. We also observed that resistance not only depended largely on the total length of the carbon fiber thread, but also on the lamination conditions. This is most likely related to the change in contact resistance conditions.The third generation of the intelligent textile is currently being tested. The concept of the parallel arrangement of a double-fiber thread was abandoned, which means that the volumetric resistance is now directly responsible for the measurement. However, the main modification is in regard to the production method. Weaving was replaced by fiber stabilization by fastening to the composite mesh matrix, and for this purpose, a glass fiber plaster-reinforcing mesh was used. The dry carbon thread was stitched to the mesh strands, as shown in The research described in this chapter is concerned with two types of sensors: the woven sensor, which was the second generation, adhered to the wooden substrate; and stabilized mesh, the latest generation, adhered to the concrete substrate. In our tests, two-component epoxy S&P Resin 55HP was used, which is an adhesive intended for fixing carbon sheets onto various types of substrate. Wet lay-up technology was also applied according to the following method: firstly, the substrate was impregnated, then the sensor was placed, and the carbon fibers were aligned and saturated with resin. The adhesive layer was leveled out with a roller.−6/°C. The precast concrete sample was made of plain concrete, and was 1000 mm long and 200 × 60 mm for the cross-section. The coefficient of the thermal expansion of concrete was equal to 12 ×10−6/°C.The models were tested after more than a month of curing the adhesive. They were stored in a stable temperature of about 20 °C and 60% humidity two hours before the test samples were placed in a climatic chamber . heating from +20 °C to +40 °C with a simultaneous humidity change from 60% to 65%,heating from +20 °C to +60 °C with a simultaneous change in humidity from 60% to 65%,cooling from +20 °C to 0 °C with a simultaneous reduction of humidity from 60% to 55%,cooling from +20 °C to −20 °C with a simultaneous reduction of humidity from 60% to 55%.Due to the preliminary nature of the research, tests for both samples were provided in four different cycles of temperature change with a small variation in humidity, which are the following:Concrete and wooden samples were tested separately. Before the next test cycle, they were stored for at least two days in the above-mentioned stable conditions to regain their natural humidity and temperature.After being placed in a climatic chamber, a control measurement was carried out using a simple ohmmeter to check initial resistance and to exclude accidental short circuits. Then, the textile sensor was connected to the Wheatstone bridge, the chamber was closed, and the test commenced. Although the climatic chamber allows for simultaneous changes in temperature and humidity, the temperature remains the priority. It should be also emphasised that increases and decreases in temperature are not linear; in the first phase there is an intense change, followed by a phase of slow adjustment to the expected temperature. This property causes the measurement paths in similar temperature ranges to vary in cycles with different limit values.During the test, the actual temperature and humidity were saved by the chamber controller, and strains simultaneously expressed by resistance changes were recorded every 60 s through a computer connected to the Wheatstone bridge. This measurement corresponded to the sum of the actual thermal deformation of the sample and apparent deformation, which expresses the wanted change in the sensor’s resistance under the influence of temperature and humidity.The influence of temperature and relative humidity on the resistance measurement must be considered simultaneously, and it should also be noted that the relative humidity itself is temperature-dependent. To simplify the interpretation of the results, measures were provided for relatively small changes in relative humidity and the results are presented only as a function of temperature. They are shown in diagrams as having a form of dependence on temperature changes on the apparent deformation measured. The measured strain consists of a real elongation of the sample as a result of thermal expansion and apparent strain resulting from the temperature sensitivity of the textile sensor. The direction of observed resistance changes is correct. The increase in temperature is indicated as elongation, while the temperature decrease is indicated as shortening. If the changes shown were purely the result of thermal deformability, then for wood the measured deformation should not exceed 0.003‰/°C, whereas for concrete it should be 0.012‰/°C; however, the obtained strains are much greater, and the temperature–strain relationship is also not linear.The sensor’s tendency to indicate apparent strain is particularly visible for rising temperatures. Considering the elasticity coefficient of carbon fibers, the measured strain > 20‰  theoretiAnother interesting observation is related to the speed of temperature changes. Heating tests from 20 °C to 40 °C and 20 °C to 60 °C, as well as cooling tests from 20 °C to 0 °C and 20 °C to −20 °C were running at different rates shown in . The patThe problem of the influence of humidity, including its condensation, also needs to be solved. The physical mechanisms of the sensor’s high sensitivity to temperature changes also require clarification. Perhaps it will be necessary to modify the concept of a developed textile sensor, such as by introducing an additional isolating layer that cuts off carbon fibers from moisture.Previous research on self-monitoring smart textiles made out of carbon fiber ,24 demonIt is thus crucial to define the relation between the readings of the structure’s deformation under the load with changing environmental conditions. Due to very few studies on this issue, the research team undertook steps to describe this phenomenon and to provide reliable tools for the determination of real strain situations in different temperatures and humidity levels.The presented tests are only preliminary and do not allow a full determination of the relationship between temperature and resistance change. However, even such limited research shows the importance and complexity of the problem. Resistance changes are not only a function of temperature changes, but also the speed of these changes or the type of substrate. Caused by increasing temperature, an indication error exceeding 5.4 GPa clearly illustrates the importance of the development of measurement methods using carbon fibers, and the development of effective methods to compensate for the changes in temperature. Authors believe that this research path may be important not only for developed smart strengthening systems, but also for other conductive fiber-based smart structures for various branches of the industry.


Graphene-based materials are widely studied to enable significant improvements in electroanalytical devices requiring new generations of robust, sensitive and low-cost electrodes. In this paper, we present a direct one-step route to synthetize a functional nitrogen-doped graphene film onto a Ni-covered silicon electrode substrate heated at high temperature, by pulsed laser deposition of carbon in the presence of a surrounding nitrogen atmosphere, with no post-deposition transfer of the film. With the ferrocene methanol system, the functionalized electrode exhibits excellent reversibility, close to the theoretical value of 59 mV, and very high sensitivity to hydrogen peroxide oxidation. Our electroanalytical results were correlated with the composition and nanoarchitecture of the N-doped graphene film containing 1.75 at % of nitrogen and identified as a few-layer defected and textured graphene film containing a balanced mixture of graphitic-N and pyrrolic-N chemical functions. The absence of nitrogen dopant in the graphene film considerably degraded some electroanalytical performances. Heat treatment extended beyond the high temperature graphene synthesis did not significantly improve any of the performances. This work contributes to a better understanding of the electrochemical mechanisms of doped graphene-based electrodes obtained by a direct and controlled synthesis process. Graphene is considered to be a promising 2D material thanks to its unique versatile properties, in particular high thermal and electrical conductivity, with many potential applications as an electrode in chemistry and as an electrocatalyst in fuel cells, field emitters, batteries, supercapacitors, and sensors .The structure and electronic properties of graphene can be tailored by heteroatom doping, in particular by incorporating nitrogen, thereby opening the band gap and transforming graphene into a semiconductor, as recently reviewed by Yadav et al.  and Xu e2O2), as well as high sensitivity and selectivity for glucose biosensing. Shao et al. [2O2 reduction was significantly reduced, and a well-defined and enhanced H2O2 reduction peak was observed around −0.2 V, demonstrating better NG electrocatalytic activity compared to undoped graphene for H2O2 sensing. Ruiyi et al. [Some authors have studied the electrochemical properties of N-doped graphene (NG) in various experimental conditions. Wang et al.  obtainedo et al.  comparedi et al.  incorpori et al.  showed ti et al.  also stuHowever, most previous studies were performed with NG synthetized using rather complex chemical routes, with limited control of the nitrogen concentration incorporated in the graphene network. Thus, the investigation of electrochemical properties of NG films obtained by more simple routes is still of great interest for the next generations of electrodes. In particular, synthesis of NG film in one step from a solid carbon source directly onto silicon electrodes has been less explored than NG films obtained by other routes, including CVD processes and various reduction processes of GO by thermal annealing, plasma treatment, hydrothermal or solvothermal reactions in the presence of a nitrogen precursor. In a previous paper , we repo2 substrate and further covered by a Ni catalytic film (150 nm). Here we optimize the previous protocol to form the NG film directly by high temperature condensation of the laser-induced carbon plasma plume in the presence of nitrogen atmosphere, onto the Si electrode previously covered by a Ni catalytic film. The structure and composition of the NG film, compared to undoped ones, were investigated by Raman spectroscopy, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (FEG-SEM). To show the interest of using NG film as an electrode for biological applications, its performance in the detection of hydrogen peroxide (H2O2) was compared to that of graphene film. H2O2 is the product of the enzymatic detection of glucose in diabetes diagnosis and its electrochemical detection is implemented in a commercial glucometer. Moreover, H2O2 is involved in different signal transduction pathways and cell fate decisions. The “redox signaling” mechanism includes the H2O2-mediated reversible oxidation of redox sensitive cysteine residues in enzymes and transcription factors, thereby altering their activities. In comparison to normal cells, cancer cells are characterized by an increased H2O2 production rate and an impaired redox balance, thereby affecting the microenvironment as well as the antitumoral immune response [Moving on from there, in the present study we investigated the electroanalytic performance of a NG-functionalized electrode obtained using the one-step PLD-TA process previously successfully used for the electroanalytical investigation of pure graphene film. In a recent review , we mentresponse . There i−6 mbar. High purity (99.99%) Ni was molten thermally in a tungsten nacelle and evaporated towards the substrate. The deposition rate was set at 1.5 nm/min to minimize residual stress in the growing film, thereby limiting film delamination. The Ni/Si samples were introduced in the PLD chamber pumped at a base pressure of 10−6 mbar, annealed at 780 °C for 30 min to increase the Ni grain size. While maintaining the temperature of 780 °C, carbon was ablated from a high purity graphite (99.9995%) target using a femtosecond laser  at a temperature of 780 °C. The Ni/Si substrates were mounted on a sample holder placed at a distance of 36 mm from the graphite target. Nitrogen gas (99.9995% purity) was introduced as a reactant gas in the vacuum chamber at a pressure of 10−1 mbar.Undoped graphene films were also synthetized using the same protocol, but without introducing nitrogen gas during carbon ablation. The ablation time was adjusted to keep both carbon and nitrogenated carbon film thicknesses equal to 40 nm. In some cases, the temperature of 780 °C was maintained for a defined period after deposition to observe the effect of longer heating periods. The procedure ended with natural cooling of the samples, before opening the vacuum chamber. In the present paper, we selected four deposition conditions to highlight the major effects due to nitrogen doping, compared to undoped films. The conditions are summarized in The morphology of the sample was observed using a field emission gun scanning electron microscope (FEG-SEM) Novananosem 200,  operated at 15 kV. Micro-Raman analyses were performed using an Aramis spectrometer , with 442 nm (2.81 eV) excitation laser focused through a ×100 objective with high aperture, ensuring the micrometric resolution of the analysis, and allowing for precise Raman mapping of the samples.2 areas were scanned, recording a Raman spectrum every 2 µm . The laser power was kept below 3 mW to avoid damaging the surface of the film. Raman components were associated with a Lorentzian fit, safe for the asymmetric G peak, which was fitted using a Breit-Wigner-Fano function. A custom fitting function was computed for all recorded spectra, and a simple linear function was added to eliminate the background. This enabled access to the exact values of the various peak positions, widths and maximum peak height intensity. XPS analysis was performed at the SOLEIL Synchrotron  on the ANTARES beam line. The ring operating conditions were 2.5 GeV electron energy, with injection currents of 500 mA and “Top-up” mode. Radiation was monochromatized using a plane-grating monochromator (PGM), which is characterized by a slitless entrance and the use of two varied linear spacing (VLS) gratings with variable groove depth (VGD) along the grating lines. The diameter of the X-ray spot impinging the surface is 140 μm and the X-ray energy was fixed at 700 eV for analysis of the graphene films. The photoemission spectra were taken with incident photon energies of 700 and 350 eV, with 190 meV and 140 meV energy resolution, respectively.For each sample, 20 × 20 µm2O2. This feature prevented further manipulation or movements of the electrodes . For this work, a saturated calomel electrode from Hach Lange  was used as the reference electrode, a planar platinum electrode (0.59 cm2) was used as the counter electrode and the nitrogen doped and undoped graphene samples were the working electrodes. The active surface of the working electrode, determined by a polyethylene terephthalate (PTFE) O-ring seal, was 0.07 cm2. This three-electrode system was connected to a Bio-Logic potentiostat VMP2 . Results were recorded using EC-Lab software (v11.27) from Bio-Logic Science Instruments. In order to characterize the electron transfer rate for the different nitrogen doped and undoped graphene samples, cyclic voltammetry was performed in a 0.5 M 1,1′ ferrocene-dimethanol solution of 0.1 M NaClO4. The scan rate was 100 mV/s. The detection of H2O2 in a non-deaerated 0.1 M phosphate buffer saline (PBS) solution (pH 7.4) was detected through linear sweep voltammetry in the anodic range from 0 to 1000 mV with a scan rate of 100 mV/s.Electrochemical measurements were carried out in a conventional one compartment three electrode cell with an internal volume of 5 mL. This electrochemical cell was designed to maintain a fixed distance between the electrodes. It was manufactured with two inlets, one for positioning the reference electrode and the other for injecting HCyclic voltammetry measurements obtained with ferrocene methanol on pure graphene and NG films are presented in E between anodic and cathodic peaks of ferrocene methanol increased with an increase in annealing time for both graphene and NG films. For the NG-0 film, in the absence of annealing following growth, the value of ∆E was close to the theoretical value of 59 mV, showing the high reversibility of the redox probe.The length of the annealing time following graphene growth had no significant effect on the intensity of the anodic peak of ferrocene methanol. The value of ∆2O2 than undoped G-60 film.The oxidation of hydrogen peroxide began at a potential value of 600 mV for both types of films, as shown in The direct observation of the graphene-covered electrode by FEG-SEM  highlighs into three components. The most intense component was centered at 284.4 eV and was attributed to sp2 hybridized C atoms in graphene. The two other ones were located at 284.9 and 285.8 eV, respectively. The component at 284.9 eV was attributed to disordered carbon (CB) and may be considered as an intermediate state between sp² and sp3 hybridizations that can be found in nano-diamond and amorphous carbon films, in agreement with References [The N/C intensity ratio deduced from the spectra was 0.01786, which is consistent with a nitrogen doping of 1.75 at %. ferences ,17,18.The component at 285.8 eV was attributed to C–N or C–O bonds ,20. Howesp2 bonding. The D band is associated with the pulsation of aromatic circles (“breathing mode”) and appears only in the presence of defects and dislocations in the graphitic matrix. The intensity ratio between the D band and G band (D/G) is thus an indication of disorder in the carbon structure. The 2D band is associated with a double resonance Raman scattering process between two aromatic circles. It appears in both graphene and graphite, and the intensity ratio of the 2D band versus the G band (2D/G) is a good indicator of the graphene-like quality of a thin film, a ratio higher than 1 being indicative of monolayer graphene. In non-defective graphene, the study of the 2D peak position and width is also a good way to count the number of layers and to identify their stacking configuration [Raman results related to the undoped and doped graphene films are shown in guration ,23. Addiguration . They emguration , which eAll the Raman spectra shown in The Raman findings are summarized in It should be noted that, in the course of this study, further post-annealing durations were tested in addition to those presented here. It was impossible to obtain any kind of pure graphene material without at least 30 min of post-deposition annealing. However, it was possible to synthesize an N-doped graphene film without post-annealing, i.e., the substrate was left to cool naturally immediately after the a-C:N pulsed-laser deposition. We would like to underline that this possibility implies an effect of the nitrogen environment on the catalysis of graphene growth by Ni, which opens the way for a more rapid fabrication of N-doped graphene. This also implies that, despite the high temperature used for graphene synthesis using annealing on a nickel catalyst, the as-deposited carbon structure may have a strong influence on the catalytic process. Additionally, it appears that lengthening the post-annealing period is an advantage with pure few-layer graphene, as the peak D/G ratio decreased and the 2D/G ratio increased when post-annealing time was increased from 60 to 90 min. This is in contrast to the fact that for N-doped graphene films, post-annealing only appeared to increase the D/G ratio, pointing to a higher number of defects.−1 [−1. The width of this peak may be associated with the distortion of the C–C sp2 bonding angle. This is to be expected due to the nanotexturing of our samples, as observed by FEG-SEM shown in −1 compared to 1582 cm−1 [All the observed peaks appear to be more intense than those of monocrystalline non-defective graphite or graphene. The full width at half maximum (FWHM) of the G peak is generally around 15 cm−1  while he582 cm−1 .These characteristics, combined with the high D versus G peak intensities ratio, are clear indicators of highly nano-crystallized graphene-like layers. The broadening of all peaks due to the nanotexturing of the substrate during annealing makes it impossible to draw clear conclusions concerning the precise number of graphene layers by studying the 2D band Full Width Half Maximum (FWHM).2O2 by the N-doped graphene films compared to undoped ones in more detail. What is the main effect among the defective nature of the N-doped graphene films, the dopant concentration or the nature/proportion of the nitrogen chemical functions in the graphene network?The objective of this section is to discuss the significant improvement of electroanalytical oxidation of HAccording to the previous section, the morphologies of all films (doped and undoped) appear to be textured, probably due to the texturing of the nickel catalyst surface caused by thermal annealing. We previously showed that the formation of nickel silicide contributed to texturing when graphene was grown on a silicon substrate covered by a Ni thin film .Likewise, all films have a defective structure, as shown by Raman investigations, and nitrogen doping at a concentration as low as 1.6 at % does not significantly influence the defective structure of graphene, which is probably inherent to the synthesis process, as already observed by Schiros et al., who compared CVD synthesis of graphene and N-doped graphene films . Moreove2O2 was observed between undoped and N-doped graphene films, and the difference was more pronounced when the heat treatment was limited during the PLD film growth in the presence of nitrogen gas. The in situ nitrogen doping process during PLD graphene growth described in the previous section, led to the formation of a few-layer graphene film containing 1.75 at % of nitrogen, with a similar proportion of pyrrolic-N and graphitic-N, and a negligible amount of pyridinic-N. According to the literature, in particular, in Wang et al. [2O2 oxidation. In particular, carbon atoms adjacent to nitrogen dopants may have a substantially positive charge density to counterbalance the higher electronic affinity of N atoms, consistent with the increased adsorption of H2O2 involved in the oxidation reaction. Such a mechanism has also been shown by density functional theory (DFT) simulating the physisorption process of H2O2 onto graphene-based surfaces [2O2 electrocatalytic oxido-reduction, the O–O bond in H2O2 was more easily broken at the surface of N-doped graphene because N doping induced the charge delocalization of graphene. With our NG-0 film, in the absence of annealing following growth, the oxidation of H2O2 began at a potential value of 600 mV. This value is rather high compared to that reported by Wang et al. (200 mV), who observed a four times higher H2O2 oxidation signal with the NG films compared to the undoped ones [2O2 oxidation signal was observed for the NG-0 film compared to the average value obtained with the undoped graphene films.However, a huge difference in electroanalytical oxidation of Hg et al.  and in Ssurfaces . Additioped ones . Our res2O2 than the number of defects, which is similar with or without N doping. Such an influence was already highlighted by Xu et al. [2O2 oxidation performance. However, our results do not follow the reactivity scale simulated by DFT by Wu et al. [2O2 oxidation reactivity occurred in the following order: pyridinic-N > Pyrrolic-N > Graphitic-N > Pristine graphene. However, it is difficult to compare DFT calculations performed with only one nitrogen-based function, even if true, with an experimental system comprised of a mixture of nitrogen-based functions embedded in a textured few-layer graphene material. Further studies are thus recommended to optimize N-doped graphene films with better control of the N-based chemical functions, in particular with distinct proportions of the various N-based functions, in order to confirm their effect as key factors for electrochemical applications of N-doped graphene films.Such high electroanalytical reactivity is generally attributed both to chemical functionalization  and to structural defects caused by nitrogen atoms. However, based on our experiments, the nature and proportions of the various N–C chemical functions certainly play a more significant role in the electroanalytical oxidation of Hu et al. . Accordiu et al. ,30, the u et al.  obtainedu et al.  who concThe electrode is covered by a few-layer defective and textured N-doped graphene film, containing 1.75 at % of nitrogen distributed in both graphitic-N and pyrrolic-N chemical functions at similar proportions.With the ferrocene methanol system, the electrode displays excellent reversibility, 60 mV, close to the theoretical value of 59 mV, and very high sensitivity for hydrogen peroxide oxidation characterized by an intensity 240 times higher than that obtained with undoped graphene synthetized using the same process, and a detection limit of 1 mM of hydrogen peroxide.These significant electroanalytical results are correlated with the amount of N doping and with the proportion of both graphitic-N and pyrrolic-N chemical functions incorporated into the defective and textured few-layer graphene film.Additional heat treatment following the deposition process does not significantly modify the nanoarchitecture of the N-doped graphene films and slightly decreases the electroanalytical performance in terms of reversibility and hydrogen peroxide oxidation performance, compared to the N-doped films obtained with no additional heating.Here we report on experimental work on the electroanalytical performance of N-doped graphene silicon-based electrodes, obtained in one step by pulse laser deposition of carbon performed in a vacuum at high temperature, in the presence of a surrounding nitrogen atmosphere. The main conclusions are the following:Further works are recommended to achieve better control of the different N-based chemical functions embedded in the graphene network and to quantify their effects on the electroanalytical performances of N-doped graphene electrodes.


A stable and highly sensitive graphene/hydrogel strain sensor is designed by introducing glycerol as a co-solvent in the formation of a hydrogel substrate and then casting a graphene solution onto the hydrogel in a simple, two-step method. This hydrogel-based strain sensor can effectively retain water in the polymer network due to the formation of strong hydrogen bonding between glycerol and water. The addition of glycerol not only enhances the stability of the hydrogel over a wider temperature range, but also increases the stretchability of the hydrogel from 800% to 2000%. The enhanced sensitivity can be attributed to the graphene film, whereby the graphene flakes redistribute to optimize the contact area under different strains. The careful design enables this sensor to be used in both stretching and bending modes. As a demonstration, the as-prepared strain sensor was applied to sense the movement of finger knuckles. Given the outstanding performance of this wearable sensor, together with the proposed scalable fabrication method, this stable and sensitive hydrogel strain sensor is considered to have great potential in the field of wearable sensors. Wearable, flexible sensors to monitor human body pressure, temperature, strain, and chemicals hold great potential in the field of bioelectronics, artificial intelligence, and soft robotics –2. AmongHydrogels, with mechanical properties like biological tissues and consisting of three-dimensional polymer networks that can retain a large amount of water, can serve as ideal vehicles for wearable devices –9. SeverIn this study, a flexible, stable, high-sensitivity, graphene-based, water/glycerol (WG) binary-solvent hydrogel (graphene/WG-hydrogel) strain sensor is designed via a two-step method. Water and glycerol are used as solvents to synthesize the hydrogel substrate with long-lasting moisture-retaining properties . The conChemicals: Acrylic acid (C3H4O2), ammonium persulfate ((NH4)2S2O8), α-ketoglutarate (C5H6O5), glycerol (C3H8O3), acrylamide (C3H5NO) and methylene-bis-acrylamide (C7H10N2O2) were purchased from Aladdin . The 2 mg/mL aqueous graphene dispersion was purchased from Tanfeng Graphene . Silver epoxy was purchased from Ted Pella .Formulation of the hydrogel: Acrylic acid, acrylamide, ammonium persulfate, methylene-bis-acrylamide and α-ketoglutarate were mixed in a beaker containing glycerol/water (1:3 v/v) binary solvent with concentrations of 40 mg/mL, 200 mg/mL, 25 mg/mL, 0.15 mg/mL, and 0.3 mg/mL, respectively, to make the monomer solution. Then the solution was mixed by magnetic stirring for 1 h. After the mixed solution was poured into the mold and irradiated by the UV light for 1 h, the hydrogel substrate was formed. The pure-water hydrogel was formulated in the same way as the binary solvent hydrogel, except that no glycerol was added.Formulation of the graphene/hydrogel: The graphene film was cast on the hydrogel film using a drop casting and drying process. 5 mL of the 2 mg/mL graphene dispersion was drop casted onto the hydrogel and put in the oven at 35 °C until the graphene was fully dried.Assembly of the strain sensor: The strain sensor was fabricated by connecting the graphene/hydrogel to a copper foil using the silver epoxy. The size of the graphene/hydrogel sensor is 5 × 30 mm.Characterization of the hydrogel and graphene/hydrogel: The mechanical properties of the hydrogel were characterized in an electronic universal testing machine . A field-emission scanning electron microscope  was used for electron microimaging. The resistance changes of the strain sensor under stretching was monitored by a multimeter (Keithley 2400 Source Meter).The graphene layer was formed on the hydrogel by directly drop casting the graphene solution onto the hydrogel and drying in an oven at 35 °C. The drying process removes the extra water in the graphene dispersion without removing the water inside the hydrogel, thus maintaining the mechanical properties of the material. The graphene layer serves to significantly increase the resistance change of the hydrogel under strain because of the spacing variations among graphene flakes. The schematic structure and a photograph of the composite electrode is shown in Besides serving to retain water in the hydrogel, the presence of glycerol also improves the mechanical performance of the hydrogel, as shown in R/R to the strain), is much higher than that of the WG-hydrogel sensor. At 25% stretching, the gauge factor of the graphene/WG-hydrogel is about 2.4, while that of the hydrogel is only about 0.59. This superior piezoresistive performance comes from two aspects: the intrinsic piezoresistive behavior of the WG-hydrogel and the electron conduction change of the graphene film under different contact conditions [The sensing performance of the graphene/WG-hydrogel composite material is shown in nditions  (spacingThe graphene/WG-hydrogel strain sensor was also used to sense flexion, another type of human movement, as shown in 2, through a simple drop casting and drying method [We have demonstrated a wearable, stable, and highly sensitive strain sensor, based on a binary solvent, graphene/WG-hydrogel composite material, synthesized using a two-step process. The long-term water-retention properties of the graphene/WG-hydrogel strain sensor can be attributed to the use of glycerol as a co-solvent. The hydrogel bonding between glycerol and water prevents water from being released from the polymer network, hence guaranteeing the long-term stability of the sensor. In addition, a graphene film is cast onto the WG-hydrogel to enhance the sensitivity of the hydrogel strain sensor. The strain sensor is demonstrated to operate in both stretching and flexuous modes, together with the ability to sense the movement of finger knuckles, suggesting the great potential of this soft and stable hydrogel-based strain sensor. In addition, the long-term moisture-retaining property of the WG-hydrogel provides an ideal substrate to cast other kinds of two-dimensional material films, such as MoSg method –18.Yield strain stress curve of the hydrogels; Cross-section SEM image of the graphene/hydrogel composite; Hysteresis curve for the graphene/WG-hydrogel strain sensor; Optical cross-section images of the graphene/WG-hydrogel composite before and after stretching.File 1Additional figures.


IHC). Expression of mRNA was assessed by ISH using the RNAScope 2.5 assay and probe CD274/PD-L1 (Advanced Cell Diagnostics) including kit provided positive and negative control probes. Brightfield whole slide images of tissues were captured. The percentage of tumour cells with PD-L1 mRNA expression (%TCmRNA) and mean punctate dots/tumour cell were determined using image analysis. Differences in RNA expression between the IHC derived TCIHC≥25% and <25% groups were assessed using t-tests. For each indication, a receiver-operating characteristic (ROC) analysis identified thresholds for patient classification using %TCmRNA and dots/tumour cell, with reference to TCIHC≥25%. Eighty-six samples were successfully tested; 3 failed due to insufficient control probe staining, 1 due to lack of tumour. Percent TCmRNA staining using RNAScope demonstrated statistical significance (at α = 0.05) in the PD-L1 high (TCIHC ≥25%) vs the PD-L1 low (TCIHC <25%) groups for NSCLC, HNSCC, and UC. The number of punctate dots/tumour cell was significantly higher in the PD-L1 high vs the PD-L1 low groups for NSCLC and HNSCC but not UC. For %TCmRNA; ROC analysis identified thresholds of: NSCLC 18.0%, HNSCC 31.8%, UC 25.8%. For dots/tumour cell, thresholds were: NSCLC 0.26, HNSCC 0.53, UC 0.45. Routine tissue fixation and processing is suitable for RNA detection using RNAScope. PD-L1 mRNA extent and level is associated with PD-L1 status determined by IHC. Threshold optimisation for %TCmRNA and mean dots/tumour cell results in high specificity to IHC PD-L1 classification, but only moderate sensitivity.Four immunohistochemistry (IHC) diagnostic assays have been approved for tumour PD-L1 protein assessment in the clinic. However, mRNA detection by in situ hybridisation (ISH) could be utilised as an alternative to protein detection. Detecting spatial changes in gene expression provides vital prognostic and diagnostic information, particularly in immune oncology where the phenotype, cellular infiltration and immune activity status may be associated with patient survival. Translation of mRNA expression to a clinically relevant cut off or threshold is challenging due to variability between assays and the detection of different analytes. These studies aim to confirm the suitability of formalin fixed paraffin embedded (FFPE) tissue sections for use with RNA ISH. A comparison of mRNA expression and protein expression may inform the suitability of mRNA as a patient selection biomarker in a similar manner to IHC and provide evidence of a suitable scoring algorithm. Ninety patient samples, thirty for each indication of non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma (UC), previously assessed using the VENTANA PD-L1 (SP263) Assay were chosen to represent a wide dynamic range of percentage tumour cell staining (TC Programmed cell death ligand 1 (PD-L1) is part of a complex system of receptors and ligands that are involved in controlling T cell activation. PD-L1 acts at multiple sites in the body to help regulate immune responses by delivering inhibitory signals to T cells through the programmed cell death 1 (PD-1) and cluster of differentiation (CD) 80 receptors. PD-L1 is a member of the B7 family of ligands that inhibit T-cell activity through binding to the PD-1 receptor . ExpressNumerous therapies have been approved or are undergoing investigation which target either PD-1 or PD-L1 and elicit anti-tumour activity . The binThe patient population may be stratified by diagnostic assays to determine those patients most likely to respond to IO therapy. Additionally, diagnostic assays may also provide data to manage the expectation of tumour response, adverse effects and tolerability. These assays are typically immunohistochemistry (IHC) assays co developed during clinical trials and at the time of writing, the only regulatory approved companion diagnostic assays for PD-L1 detection are IHC based , 9. In aMultiple IHC PD-L1 assays have been developed for use with immunotherapies; each with different cut-offs and scoring algorithms. The ability to interchange assays facilitates PD-L1 testing for patient eligibility. Different assays show varying concordance, for example Ratcliffe et al  demonstrin situ hybridisation (ISH). ISH has been shown to be an alternative to IHC, for example for the detection of PD-L1 [HER2 amplification [MET amplification [in situ offers an insight into the heterogeneity of tissue. Morphology of tissue may impact RNA expression and could possibly vary in neighbouring cells. Quantifying the amount of gene expression may provide additional sensitivity and specificity over other assays, offering greater predictive power for patient outcome and/or stratification, potentially increasing the patient population that will respond to targeted therapy [Assays which detect alternative molecules to protein have been investigated across pathologies and in different indications including those which measure mRNA or DNA by of PD-L1 , 15 and fication  PTEN [17fication . These a therapy , 19.RNAScope is a commercially available, fully automated, ISH assay for the detection of RNA in a variety of sample types including formalin fixed paraffin embedded tissue , 21. TheAdditionally, RNA ISH has been used to retrospectively investigate clinical response and the extent of RNA expression in comparison to IHC . AlthougScoring algorithms for PD-L1 status using RNAScope have been previously proposed. For example, greater than 10 punctate dots per cell  or greatHere in, PD-L1 RNA expression determined using RNAScope and protein expression determined using a commercially available IHC assay (VENTANA PD-L1 (SP263)) are compared. Data are used to identify a novel threshold to classify tissue as PD-L1 high/low using RNAScope, providing evidence of a potential scoring algorithm that has been derived from a commercially available IHC assay.IHC). This tissue set has previously been assessed using other PD-L1 IHC assays such as Dako PD-L1 IHC 22C3 pharmDx and PD-L1 IHC 28–8 pharmDx assays [Ninety (90) commercial clinical cases, thirty (30) for each of NSCLC  HNSCC and UC , previously assessed using the VENTANA PD-L1 SP263) Assay  were chosen to represent a wide range of percentage tumour cell membrane staining  on a Leica Rx instrument  as described previously . BrieflyACD positive control probe (Peptidylprolyl Isomerase B (Cyclophilin B) (PPIB) and negative control probe (B. subtilis gene dihydrodipicolinate reductase) (dapB) were run on each tissue prior to testing with CD274/PD-L1 probe. Tissue passed quality control if the average number of punctate dots per cell throughout the entire sample were greater than five using the PPIB probe and zero using the dapB probe.mRNA) and the level of expression (mean punctate dots/tumour cell) using RNAScope were determined.Bright field images were captured using an Aperio AT2 digital slide scanner  and ISH scores were generated using HALO (Indica Labs) image analysis software. Tumour regions of interest were annotated by a board-certified pathologist prior to image analysis in order to standardise tumour areas selected for comparison. Regions of interest totalled 25–50% of the available tumour across the tissue section, the range of cells analysed per tissue section were between 468 to 190,645. Tumour islands were identified and separated from prominent tissue associated stroma. The percentage of tumour cells with any PD-L1 mRNA expression  staining was not assessed. Samples were read by a single pathologist trained by the manufacturer in a Clinical Laboratory Improvement Amendments (CLIA) program–certified laboratory . Equal numbers of PD-L1 high and PD-L1 low cases from each indication were selected for RNAScope staining based on the 25% PD-L1 TCIHC cut off. Representative images of IHC and RNAScope staining are shown in All samples had previously been stained using the automated VENTANA BenchMark ULTRA platform using the VENTANA PD-L1 (SP263) rabbit mAb assay according to package inserts. Tissue assessment was based on tumour cell membrane staining in all indications. Cases with TCmRNA and dots/tumour cell between PD-L1 high (TCIHC ≥25%) and PD-L1 low (TCIHC <25%) groups were assessed using t-tests (Microsoft Excel 2016).Differences in %TCmRNA and dots/tumour cell, with reference to TCIHC ≥25%.For each indication, strip charts and a receiver-operating characteristic (ROC) analysis  were prNinety (90) FFPE cases from NSCLC, HNSCC and UC were assessed for analysis with RNAScope. Eighty-six of the tumours showed PD-L1 staining with RNAScope. One UC case was not analysed due to the absence of tumour. Two NSCLC cases and one HNSCC case were not analysed due to insufficient/absent staining using RNAScope positive control probes (QC fails).Twenty-eight NSCLC cases were successfully stained using both SP263 and RNAScope assays. Twenty-nine HNSCC cases were successfully stained using both SP263 and RNAScope assays. Twenty-nine UC cases were successfully stained using both SP263 and RNAScope assays.mRNA staining for PD-L1 mRNA ranged from 1.31% to 75.99% in NSCLC, from 4.55% to 58.63% in HNSCC and 0.19% to 89.8% in UC (The %TC8% in UC . The dot8% in UC .mRNA was significantly higher (at α = 0.05) in the TCIHC≥25% vs <25% groups for NSCLC (38.7 vs 13.1% p = 0.0014), HNSCC (35.1 vs 20.2 p = 0.0040) and UC (31.4 vs 12.4% p = 0.030).%TCIHC≥25% vs <25% groups for NSCLC (0.99 vs 0.24 p = 0.0083) and HNSCC (0.73 vs 0.32 p = 0.0023) but not UC (0.25 vs 1.0 p = 0.12). Data shown in Dots/tumour cell was significantly higher in the TCmRNA and average dots per cell using RNAScope. Analytical cut off determination was based on the highest sensitivity and specificity of the RNAScope assay against IHC (SP263) using a 25% tumour cell staining cut off. For %TCmRNA, the ROC analysis identified thresholds of: NSCLC 18.0%, HNSCC 31.8%, UC 25.8%. For dots/tumour cell, ROC analysis identified thresholds of: NSCLC 0.26, HNSCC 0.52, UC 0.45  and expression level (the amount of staining per tumour cell) demonstrating positive association with PD-L1 status as determined by IHC. The %TCTissue for this study represented a variety of indications and sample acquisition methods which routine laboratories may encounter. mRNA was quantifiable in 96% of cases, demonstrating, as has been shown previously, that routine tissue processing is suitable for the preservation of RNA . mRNA waThe VENTANA PD-L1 (SP263) rabbit mAb assay was used in this study. IHC assays such as Dako PD-L1 IHC 22C3 and 28–8 assays have been shown to demonstrate good concordance with each other , 12, 25;mRNA and mean dots/tumour cell, results in high specificity to IHC PD-L1 classification, but only moderate sensitivity. The RNAScope assay, if compared to VENTANA PD-L1 (SP263) rabbit mAb assay (as a gold standard), would select a marginally different PD-L1 high patient population using the cut offs derived here. For NSCLC, %TCmRNA threshold at 18% and dots/tumour cell cut off at 0.26, 85.7% of PD-L1 high IHC cases would also be PD-L1 high by RNAScope and 78.6% of PD-L1 low IHC cases would also be classified as PD-L1 low by RNAScope. For HNSCC, using %TCmRNA cut off at 31.8% or 0.5 dots per cell, 60% of PD-L1 high IHC cases would also be PD-L1 high by RNAScope. Using these cut off values RNAScope showed 100% or 93% specificity for %TCmRNA and dots per cell respectively. For UC using %TCmRNA cut off 25.8% or 0.45 dots/tumour cell, 53.3% of PD-L1 high IHC cases would also be PD-L1 high by RNAScope and 92.9% of PD-L1 low IHC cases would also be classified as PD-L1 low by RNAScope. The moderate sensitivity of RNAScope compared with IHC results in selection of a larger PD-L1 low patient population, the clinical response of which is unknown. No data regarding patient response was available in this study, and further clinical validation of mRNA detection using RNAScope to select patients is required.Threshold optimisation for %TCThese data were derived using the anti-human PD-L1 rabbit mAb (SP263), PD-L1 high staining was defined as ≥25% tumour cell staining at any intensity. Alternative thresholds in different indications may yield different results . EvidencRNAScope could be used to aid the identification of immune cells by staining specific immune cell markers, such as CD4 and CD8. Combining RNAScope with IHC could potentially enable better patient stratification and therefore efficacy to treatments which target immune checkpoint blockade. RNAScope has been developed previously as a dual stain utilising detection of RNA and protein . ChallenAlternatively, a multiplex assay targeting PD-L1 transcripts and protein by RNAScope and IHC could be used to identify subsets of patients, for example those that are PD-L1 IHC low, but mRNA expression high, may respond differently to PD-L1 targeted therapies. Additionally, a multiplex approach will reduce the tissue requirements of a diagnostic assay on small, precious clinical samples .Here, RNA expression was measured using a combination of a commercially available assay and image analysis software. Quantifying the amount of RNA across a whole tumour section in individual tumour cells is however challenging. Manually quantifying mRNA expression requires an extensive amount of time and may be prone to error due to the complexity associated with the number of tumour cells present in a tissue section and the relationship with the surrounding stroma. One approach to simplify analysis is to enumerate a small number of representative cells, this approach may not be ideal, potentially excluding a large amount of useful data. Alternatively, as employed in this study, RNAScope may be exploited in combination with image analysis software, allowing large numbers of tumour and immune cells to be analysed. Image analysis may prove particularly useful to standardise the analysis of immune cells, especially if a multiplex assay can be employed as described above. Image analysis will also enable any heterogeneity within the expression profile to be investigated and analysed. Accurate determination of PD-L1 staining in large numbers of tumour cells, identification of immune cells and identification of heterogeneity may be useful to direct patient treatment options especially in cases which are equivocal or borderline .Data presented here was quantified using commercially available image analysis software. Analysis is based on predefined rules and is driven by a fixed algorithm. The use of machine learning to aid clinical diagnosis has been explored using immunohistochemistry in breast cancer . RNAScopWe have shown detection of PD-L1 RNA in commercially available clinical samples using RNAScope demonstrates high specificity and moderate sensitivity with protein detection using a commercially available PD-L1 IHC assay (Ventana SP263). Both assays would select marginally different patient populations. Due to the moderate sensitivity of RNAScope compared to IHC, RNAScope identifies a population of cases across NSCLC, HNSCC and UC as PD-L1 high that would not be PD-L1 high by IHC. The response of these patients to treatment with PD-L1 immunotherapy is unknown. The thresholds presented here for RNAScope are for the first time derived using a commercially available IHC assay as a reference but further validation with patient outcome data is required.


Despite the large number of performed studies, the etiology and pathogenesis of sarcoidosis still remain unknown. Most researchers allude to the possible autoimmune or immune-mediated genesis of the disease. This review attempts an integral analysis of currently available information suggesting an autoimmune genesis of sarcoidosis and is divided into four categories: the evaluation of clinical signs described both in patients with sarcoidosis and “classic” autoimmune diseases, the role of triggering factors in the development of sarcoidosis, the presence of immunogenic susceptibility in the development of the disease, and the analysis of cellular and humoral immune responses in sarcoidosis. Studying the etiology and pathogenesis of sarcoidosis will improve diagnostic procedures as well as the prognosis and patients' quality of life. Sarcoidosis is a multisystem granulomatous disease of unknown origin with predominant lung involvement, variable clinical course, and no universally accepted treatment algorithm , 2. The Recently, much attention has been paid to the study of the etiology and pathogenesis of sarcoidosis and the role of autoimmunity in its development and progression. In addition to genetic predisposition, triggers such as infection, inorganic materials, and environmental factors are likely to play a role, which leads to the possible development of an autoimmune response in the disease , 3.Sarcoidosis has a wide variety of clinical phenotypes wherein many of them remind “classic” autoimmune diseases. About half of the patients have no symptoms, while in severe clinical cases, sarcoidosis can lead to a failure of the internal organ functions with the development of fibrosis and pulmonary hypertension. The most common clinical symptoms of pulmonary sarcoidosis are a dry cough, shortness of breath, and chest pain, which are described in more than 50% of patients. Up to 30% of cases include fever, unexplained weight loss, and chronic fatigue , 2. ExtrPulmonary sarcoidosis may coexist with other autoimmune diseases, which suggests the possibility of a common pathogenesis and genetic predisposition. Most often, cases of sarcoidosis associated with Sjogren's syndrome are described, where patients have signs of both diseases, e.g., epithelioid granulomas in their internal organs as well as anti-Ro and anti-La autoantibodies as well as lacrimal and salivary gland dysfunction. Sarcoidosis and Sjogren's syndrome are both associated with the HLA-DR3 genotype and elevated levels of CD4+ lymphocytes , 6.In some cases, a combination of systemic lupus erythematosus (SLE) and sarcoidosis is observed. As a rule, SLE debuts as a primary disease where over time non-caseating granulomas in the skin and lungs are detected . These cThe combination of sarcoidosis with Crohn's disease, antiphospholipid syndrome, idiopathic pulmonary fibrosis, and primary biliary cirrhosis is also described . When saOne of the most important evidence of the autoimmune inflammation in sarcoidosis is the formation of granulomas, mainly in the lungs and the mediastinal lymph nodes as well as in the skin and liver of patients. The formation of granulomas of both infectious and autoimmune nature is based on the aggregation of hypertrophic macrophages. In infectious granulomatosis, such as tuberculosis, a pronounced activation of Th1 lymphocytes and M1 macrophages which then progress to the M2 phenotype is described. In sarcoidosis the opposite situation is observed: the M2 (anti-inflammatory) macrophage phenotype predominates in granulomas, which represents paradoxically reduced immune response. When studying the molecular mechanisms underlying the dysfunction of macrophages, there is growing evidence of the role of the mTOR pathway in granuloma formation. Linke and Wilson et al. in their studies showed that activation of rapamycin (mTOR) complex 1 (mTORC1) in macrophages in mice leads to the progression and development of granulomas. It has also been shown in people with a progressive sarcoidosis , 11.After processing of environmental immunogenic antigens by mononuclear antigen-presenting cells, the antigen is presented to T-effector cells. Further activation of immune cells occurs with the help of various chemokines, such as CCL5 (for Tx1) and CCL2 (for Tx2). Recently, a lot of attention has been paid to the role of T17 lymphocytes in the pathogenesis of sarcoidosis, a subgroup of CD4+ lymphocytes expressing IL-17A, which exhibit pro-inflammatory or anti-inflammatory properties in response to the local inflammatory milieu. It has been shown that in sarcoidosis granuloma macrophages express CCR20, causing the attraction of Tx17, and the expression of IL-23 causes a significant increase in the concentration of IL-17A , 12. TheConsidering the possible autoimmune nature of sarcoidosis, it is necessary to discuss the possibilities of treatment with immunosuppressive therapy. Corticosteroids for patients with sarcoidosis are considered as the treatment of choice despite many possible contraindications and side effects , 14. If in vitro destruction of cells carrying specific antigen. Indirect evidence includes detection of the corresponding antigen in humans or its equivalent in animals and reproduction of essential features of the disease by immunization. Circumstantial evidence includes association with other autoimmune diseases, lymphocytic infiltration, statistical association with specific HLA haplotype, and positive response to immunosuppression.Despite a large number of studies on sarcoidosis, its etiology is still not fully understood. To consider the possible autoimmune nature of sarcoidosis, the criteria proposed in 1993 by N.R. Rose and C. Bona can be used . The search was performed with keywords: Sarcoidosis, autoimmune reactions, autoimmunity, antibodies, and HLA genotype.Mycobacterium tuberculosis and Propionibacterium acnes are most widely studied , the most pronounced associations being with sarcoidosis, Sjogren's syndrome, and systemic scleroderma after silicone implantation . These cOne of the possible triggering factors that increases the incidence of sarcoidosis is inorganic dust. In epidemiological studies, the mechanism and specific components of dust causing granuloma formation were not demonstrated, however, an increase in the incidence of sarcoidosis after the collapse of the World Trade Center on September 11, 2001 was recorded , 37. WheThere is some evidence in scientific literature of the effect of printer or copier toner ink on the development of sarcoidosis. For example, Armbruster et al. described a 39-year-old non-smoker who, after 18 months working at a newspaper agency, had intrathoracic lymphadenopathy and disseminated changes in the lungs . Histoloin vitro experiments, experiments on animal models, and clinical observations have shown the role of environmental factors , infectious agents  in the development of sarcoidosis. These factors can play an important role in the development of the disease  and CD68, which was different for some cytoskeleton proteins  .*0301 genotype  are presented on the membrane surfaces of all cell types, with the exception of trophoblast cells, and present antigens localized in the cells' cytosol of T lymphocytes. The MHC class II molecules  are predominantly expressed in the antigen-presenting cells, which represent antigens absorbed from the extracellular space by endocytosis. Presentation of the body's own antigens in association with MHC I or MHC II and their subsequent recognition by autoreactive cytotoxic T lymphocytes or T-helper cells, respectively, is a key mechanism for triggering autoimmune reactions .*04/*15 alleles are more likely to develop cardiovascular sarcoidosis, and the HLA-DRB1*04 alleles are more likely to develop uveitis  and class II . Specific HLA genotypes have been identified for the various forms of the disease, the spectrum of the affected organs, as well as ethnic characteristics. For example, it was shown that carriers of the HLA-DRB1 uveitis . The HLA*11:01 increased the risk of disease in both ethnic groups, whereas HLA-DRB1*12: 01/15: 03 was more common for African Americans, HLA-DRB1*15: 01 / 04:01—for the Caucasian race . The role of this gene in the pathogenesis of sarcoidosis is not yet known, but its relationship with sarcoidosis among African Americans, and Europeans has been shown .The described immunological and immunogenetic disorders do not arise independently, but under the influence of certain triggering factors that activate and trigger the pathogenesis mechanism of the autoimmune response. Currently, this relationship is described in many studies.The main role in the pathogenesis of autoimmune diseases is played by T-cell immunity represented by the 1, 2, 17 Th subtype of T helpers, regulatory T-cells, and also T-killers. It is believed that disturbance of the Th17 ratio stimulates the T regulatory cells, which play a role as suppressors of immune responses, and can lead to autoimmune inflammation characterized by the presence of autoantigen-specific T and B lymphocytes producing autoantibodies .In various studies on sarcoidosis, disturbances were found in T-cell and B-cell immune responses. It is believed that the triggering factor for inflammation development in sarcoidosis is the contact of the foreign antigen with antigen-presenting cells leading to activation of T and B lymphocytes, which migrate to the inflammatory foci. It is observed, that due to the long persistence of the antigen, macrophages undergo epithelioid differentiation, lymphocytes constantly migrate to the center, thus, granulomas are formed , 57.Considering that lymphocytes play the main role in granuloma formation, it can be said that the disturbance of cellular and humoral immune response is observed in sarcoidosis.As has been noted above, a distinctive feature of sarcoidosis is the formation of non-caseous granulomas, which include various immune competent cells. Thus, in the central part of such granulomas, macrophages or multinucleated giant cells and CD3+ CD4+ T-helpers predominate as a result of their fusion, whereas cytotoxic CD3+ CD8+ T-lymphocytes, regulatory T-cells, fibroblasts, and B-lymphocytes predominate in the peripheral part of the granulomas , 57. TheThis circumstance allowed us to treat sarcoidosis primarily as an infectious disease. However, the description of a fundamentally new population of T-helper cells—T-helper 17 (Th17) as well as its separate highly specialized subtype—Th1/Th17 or Th17.1 cells capable of producing both IFNγ and IL-17A at the same time, suggests the autoimmune nature of the disease , 59. FurIn patients with sarcoidosis, an increase in Th17 was found in the peripheral blood and BAL, while the number of T regulatory cells in BAL was reduced and increased in the peripheral blood . Also, iCytotoxic T-lymphocytes, along with Th, effectively produce key pro-inflammatory cytokines (TNFα and IFNγ), ensuring their high concentration at the sites of granuloma formation, which activates macrophages and attracts additional cells from the circulation . At pres*03 proteins was detected  . Later, detected . When stted  and, correspondingly, Ig-producing B-cells in the lung tissues, while there are various ratios of B-cell types , 70. In The earliest data on autoantibodies in sarcoidosis were based on the results obtained from the patients diagnosed with sarcoidosis and various chronic liver diseases, in whom antimitochondrial antibodies were identified. Similar results were obtained in 2005 when examining patients with sarcoidosis and primary biliary cirrhosis. The authors find it difficult to name the underlying cause of an autoimmune response because the presence of antimitochondrial antibodies is characteristic of autoimmune liver damage –77.Since sarcoidosis combines with other autoimmune diseases, the Weinberg group analyzed the presence of anti-nuclear antibodies and autoantibodies to double-stranded DNA to determine whether these markers could be used as predictors of disease development, such as systemic lupus erythematosus. However, antinuclear antibodies were detected in only one third of the patients, whereas antibodies to double-stranded DNA were in two patients, and therefore, these serological markers are not diagnostically significant .In 2014, the S. Kobak group began studying autoantibodies specific for rheumatoid arthritis in patients with sarcoidosis. Of the 42 patients with sarcoidosis, only seven had a higher concentration of the rheumatoid factor, two of them had antibodies to citrullinated cyclic peptides. The anti-nuclear antibodies were detected in 12 patients. However, due to the low specificity of the detected antibodies, these serological indicators cannot be used as biomarkers of sarcoidosis , 80.*03 genotype was revealed  are the first in the cascade of inflammatory reactions. It is believed that with prolonged contact of foreign antigens with the toll-like receptors of antigen-presenting cells, macrophage activation and epithelioid differentiation occur. Macrophages begin to synthesize pro-inflammatory cytokines such as nuclear factor (NF)-kB, TNF-a, IL-1, which leads to the activation of the acquired immune system. B-lymphocytes and T-lymphocytes actively migrate to the focus of inflammation. T-cells undergo differentiation into T-helpers of the 1st, 2nd, 17th type, and regulatory T-cells, depending on secreted cytokines. The inability to quickly and efficiently eliminate a foreign antigen leads to the formation of granulomas , 21. HowAutoinflammatory diseases are characterized by chronic systemic inflammation caused by activation of innate immunity  and manifested in lesions mainly of the musculoskeletal system, skin, and mucous membranes . This grAs shown above, the development of sarcoidosis is associated with a large number of genes, in particular with class II HLA genes. Moreover, with sarcoidosis, autospecific T and B cells that produce various autoantibodies are found. These facts do not allow to suggest an exclusively auto-inflammatory theory of sarcoidosis. Perhaps sarcoidosis begins as an auto-inflammatory disease in which the activation of adaptive immunity occurs with the appearance of autospecific T and B cells that produce autoantibodies to a specific antigen .- the presence of various autoantibodies in the blood of patients;- association with other autoimmune diseases ;- association with the HLA genotype ;- lymphocytic organ infiltration (the presence of sarcoid granulomas);- response to immunosuppressive therapy.According to the criteria for autoimmune diseases proposed by Rose and Bona , in sarcAt present, there is lack of evidence on the possible autoantigen in sarcoidosis, which induces the formation of sarcoid granulomas, antibodies, and T-specific lymphocytes in animal models to confirm the autoimmune origin of sarcoidosis. Vimentin appears to be the most likely antigen, against which the Grunewald group has detected both antibodies and vimentin-specific T cells.The data analyzed in the review suggest the presence of autoimmune/inflammatory syndrome induced by adjuvants described in 2011 by Shoenfeld and Agmon-Levin . The synThe described major  and minor (immunogenetic) ASIA criteria enable us to assume the autoimmune nature of inflammation in sarcoidosis. Analyzing the proposed algorithms to detect autoimmune diseases, a unified approach to assess the possible autoimmune origin of sarcoidosis can be proposed: Identification of trigger factors affecting the development of sarcoidosis; evaluation of immunogenetic susceptibility to the development of the disease; analysis of the immunological features in sarcoidosis; analysis of the clinical features of autoimmune diseases.The analysis of the current data does not exclude the auto-inflammatory nature of the occurrence and development of sarcoidosis. Thus, setting the final point in the etiology and pathogenesis of the disease is not yet possible.AS: main author. AM, NB, YZ, IK, GE, LS, and LC: analysis of literature. VM: text translation. PY: coordinator of the project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


OBJECTIVES/SPECIFIC AIMS: Long interspersed element-1s (L1s) are autonomous, mobile elements that are able to copy and insert themselves throughout the genome with their own reverse transcriptase and endonuclease. These elements make up 17% of the human genome with over 500,000 copies, though the vast majority of L1s are defective with only a few dozen potentially responsible for L1 activity. Full-length L1s have the potential to contribute to mutagenesis through random insertion and increased genetic instability. Here we set out to study L1 expression at the specific loci level in bone marrow-derived stem cells (bmSCs) and adipose-derived stem cells (ASCs) and compare the levels of expression from ASCs from donor patients who are young and lean, obese, and old. Our hypothesis is that L1-related damage may contribute to mutation and inflammation that alters the function of these stem cells throughout the life of an individual. METHODS/STUDY POPULATION: ASCs and bmSCs were isolated from patient donors. The following samples were collected: ASCs from 3 young (under the age of 59) and lean (BMI<30) patients, ASCs from 3 older patients (over the age of 59), ASCs from 3 patients with BMI>30, and bmSCs from 4 young and lean patients. Cytoplasmic RNA from the cell populations was isolated and sequenced by RNA-Seq from the cell populations. Using our recently developed bioinformatics pipeline, we set out to quantify L1 expression and identify the few culprit L1s at specific loci that are actively transcribing to RNA in the ASC and bmSC samples. RESULTS/ANTICIPATED RESULTS: Here we provide proof of concept with the application of this novel method in characterizing full-length expressed L1s at the specific loci level in ASCs and bmSCs. We identified L1 loci that are commonly expressed in these cell types and observed an increase in L1 expression in the obese and old ASC cells compared with the young, lean ASCs and bmSCs. DISCUSSION/SIGNIFICANCE OF IMPACT: ASCs hold the promise of broad application in the biomedical field including regenerative treatment. There are reports that ASCs cultivated from older and obese donors are less effective in regenerative treatments. By demonstrating an increased expression of the mutagenic L1 element in ASCs from obese and old donors, this study provides further evidence suggesting the preferable use of ASCs from young and lean donors for regenerative therapies. These studies will also help us to understand the potential contribution of L1 expression to loss of stem cell function during the aging process.


Gastroesophageal reflux disease (GORD) is highly prevalent in idiopathic pulmonary fibrosis (IPF) and may play a role in its pathogenesis. Recent IPF treatment guidelines suggest that all patients with IPF be considered for antacid therapy. However, emerging evidence suggests that antacid therapy does not improve IPF patient outcomes and may increase the risk of pulmonary infection.Using prospectively collected data from the Australian IPF Registry including use of antacid therapy, GORD diagnosis and GORD symptoms, the relationship of these GORD variables to survival and disease progression was assessed. The severity of GORD symptoms using the frequency scale for symptoms of GORD (FSSG) and its relationships to outcomes was also assessed for the first time in an IPF cohort.n = 384; 65%), though fewer had a diagnosis of GORD  and typical GORD symptoms were even less common . The mean FSSG score was 8.39 ± 7.45 with 43% (n = 251) having a score > 8. Overall, there was no difference in survival or disease progression, regardless of antacid treatment, GORD diagnosis or GORD symptoms.Five hundred eighty-seven (86%) of the 684 patients in the Australian IPF Registry were eligible for inclusion. Patients were mostly male (69%), aged 71.0 ± 8.5 years with moderate disease . Most patients were taking antacids (Neither the use of antacid therapy nor the presence of GORD symptoms affects longer term outcomes in IPF patients. This contributes to the increasing evidence that antacid therapy may not be beneficial in IPF patients and that GORD directed therapy should be considered on an individual basis to treat the symptoms of reflux. Despite recent treatment advances , 2, idioCurrent IPF treatment guidelines make a conditional recommendation for considering antacid therapy for all IPF patients irrespective of GORD symptoms . This waUsing data from the Australian IPF Registry, we explored: 1) the use of antacid therapy in our IPF population; and 2) the presence of GORD symptoms and disease, on disease progression and survival. For the first time in IPF, we also utilised the frequency scale for symptoms of GORD (FSSG) which was developed to predict the presence of endoscopic features and severity of oesophagitis resulting from GORD . If clinAll participants from the Australian IPF Registry who had completed questionnaires regarding co-morbidities, treatment and reflux symptoms were included in the study. The Australian IPF Registry is a prospective national registry which was established in 2012 for patients with IPF across Australia. Demographic, questionnaire and objective investigational data are collected at baseline and six-monthly during follow up. Details of the Registry have been published previously . The datDiagnosis of GORD (Yes/No)Typical GORD symptoms of heartburn, reflux or sour taste in mouth after eating (Yes/No)List of current medicationsFrequency scale for the symptoms of GORD (FSSG) Table . A symptIn the baseline Registry questionnaire, all Registry participants are routinely asked about the following:p value < 0.05 are reported as significant.Results are presented as mean ± standard deviation (SD), median and interquartile range (IQR) or n (%) as appropriate. Comparisons between groups were made using Student’s t test or chi squared. An unstructured, linear mixed model with random intercepts and slopes was used to calculate the annual decline in FVC % predicted. Survival analysis was performed with Cox proportional hazards and Kaplan Meier methodology. All results with a n = 406; 69%) and ex/current smokers   at the time of entry into the Registry, with most receiving proton pump inhibitors (PPIs) , and fewer receiving Histamine-2 receptor antagonists (H2RA)  and a small proportion on both PPIs and H2RAs . Of those receiving antacid treatment, 193 patients (50%) had significant symptoms (FSSG> 8) while 191 patients (50%) reported less significant reflux symptoms (FSSG≤8). The self-reported prevalence of GORD was 41.4% (n = 243) but only 29.1% (n = 171) reported typical symptoms of “heartburn, reflux or sour taste in mouth after eating”. The mean FSSG score was 8.39/48 (SD 7.45), with 43% (n = 251) patients having a FSSG score > 8.Of the 684 patients in the Registry at the time of this analysis, 587 (86%) patients had completed the baseline questionnaire. The characteristics of this population were typical for IPF with most patients being older (71.0 ± 8.5 years), mostly male , 240 (40.9%) patients died and 33 (5.6%) had a lung transplant.There was no difference in the annual fall in FVC %predicted demonstrated, regardless of whether patients were receiving antacid therapy (compared to those not on therapy), or had a GORD diagnosis (compared to those without a diagnosis of GORD), or had significant GORD symptoms (FSSG> 8) (compared to those without significant GORD symptoms (FSSG ≤8) Table .Table 3AOn univariable analysis for mortality, there was also no difference between patients who were receiving antacid treatment compared to those not receiving antacid treatment Table . There wp = 0.928), adjusting for age, gender, smoking, FVC %predicted and DLco %predicted. Increasing age, reducing FVC %predicted and DLco %predicted levels, were independent predictors of worse overall survival.On multivariable analysis, there was no difference in survival between antacid therapy groups HR = 1.02; 95% CI 0.72–1.43; p = 0.415) between those with significant GORD symptoms (FSSG> 8), compared to those without (FSSG≤8) significant GORD symptoms  compared to patients with other interstitial lung diseases (ILDs) (n = 5/20) and those with no ILD (n = 0/16) [The conflicting results surrounding antacid therapy in IPF may reflect the contribution of non-acid reflux in the pathogenesis of IPF. While it has been postulated that micro-aspiration of acid results in lung injury, refluxate in GORD also contains other potentially harmful substances such as bile salts, enzymes and bacteria . A study = 0/16) , while o = 0/16) . The refp = 0.28), and has been ascribed possibly to the small sample size [Anti-reflux surgery would prevent the reflux of both acid and non-acid refluxate and thus a prospective, randomised controlled trial to compare the decline in FVC between surgery and non-surgery groups has been performed. In this study of 58 IPF patients, patients who had surgery had a slower decline in FVC over 48 weeks (50mLs) compared to those who did not have surgery (130mLs). However this difference did not reach statistical significance (ple size . With a ple size .Another major problem facing the investigation of the relationship between GORD and IPF is the subjective nature of the GORD definition. While GORD is defined as the presence of troublesome symptoms and/or complications , 27, “trDue to the retrospective nature of this study, there are several limitations. Firstly, the diagnosis of GORD was self-reported and may under or overestimate prevalence. For this reason, we looked at many GORD related variables to assess the impact of GORD and GORD treatment. Secondly, all antacid use was self-reported at baseline and analysed using an intention to treat approach. This approach does not reflect ongoing use nor quantify total exposure, which may be of clinical significance. In a retrospective study of 786 IPF patients, the use of PPIs for > 4 months was associated with reduced mortality whereas use for 2 or 3 months was not . The intWhile further, robust randomised controlled trials are still needed, this study adds weight to the gathering evidence that antacid therapy may not be beneficial in IPF patients and that reflux directed therapy should be considered on an individual basis. While the severity of reflux symptoms does not predict IPF outcomes, it may help select patients who will benefit from GORD treatment for management of symptoms and the prevention of oesophageal injury. Our study also highlights the difficulty in defining GORD and highlights the need to standardise the GORD definition used in future prospective randomised studies if evidence of treatment benefit is to be accurately assessed.