ID/IG) from ca. 0.09 to 0.22. The translocations of anionic and cationic monomers through graphene are significantly slower due to chemical interactions of monomers with the graphene defects. Interestingly, if micropatterned initiator-monolayers are used, the translocations of anionic monomers apparently cut the graphene sheet into congruent microscopic structures. The varied interactions between monomers and graphene defects are further investigated by quantum molecular dynamics simulations.Mass transport through graphene is receiving increasing attention due to the potential for molecular sieving. Experimental studies are mostly limited to the translocation of protons, ions, and water molecules, and results for larger molecules through graphene are rare. Here, we perform controlled radical polymerization with surface-anchored self-assembled initiator monolayer in a monomer solution with single-layer graphene separating the initiator from the monomer. We demonstrate that neutral monomers are able to pass through the graphene (via native defects) and increase the graphene defects ratio (Raman  Translocation of larger molecules through graphene holds potential for molecular sieving, however it is rarely observed. Here, the authors show the radical polymerization of vinyl monomers via their translocation through a single layer graphene to a monolayer initiator and additionally study the monomer-graphene interactions. In fact, a pristine graphene sheet is mostly impermeable to all atoms and molecules due to the unfavorable energy barriers of its closely spaced carbon atoms6. Even graphene prepared by chemical vapor deposition (CVD), which is expected to have Stone–Wales defects, is theoretically demonstrated to be impermeable to helium under ambient conditions7. As such, research has shown that graphene can be used as an effective barrier to oxidation of metal surfaces under certain conditions10, as well as barrier for the deposition of self-assembled monolayers11. However, in another scenario, ions and molecules have been observed to be able to transport through single-layer graphene (via intrinsic defects) promoted by external pressure or concentration gradient14. More interestingly, Geim and colleagues15 recently showed that, upon electronic potential, pristine graphene can be highly permeable to thermal protons under ambient conditions, and Geiger and colleagues16 reported a reversible proton transfer through graphene from the aqueous phase silanol groups of a substrate surface, which correlate principally to the pioneer work of voltage-driven ionic transport across suspended CVD graphene by Golovchenko and colleagues17 in 2010. These studies suggest that the permeability of single-layer graphene could be significant  if mass-transport is driven by an external force19.Graphene and other two-dimensional materials are promising candidates for next-generation separation membranes owing to their atomic thickness20: Can possibly also organic molecules such as monomers pass through single-layer CVD graphene? If so, what happens to graphene? Is the size and/or the charge of the transported monomers of importance? Since atomic-thin graphene is transparent to van der Waals21 and Coulomb forces22, it should be possible that the graphene-covered radical initiator monolayer24 can still interact with reactants  on the other side of graphene. Meanwhile, the living nature of controlled radical polymerization (CRP) requires a continuous transport of the monomers through the graphene membrane and steadily consumes monomers near the graphene sheet to surface-bound polymer brushes In this work, we show that neutral monomers of various sizes are able to smoothly pass through monolayer CVD graphene under the driven force of radical initiator in polymerization, although the native defects of graphene are largely increased due to the translocation. In contrast, the passage of charged monomers is severely interrupted by the graphene. Notably, the translocation of anionic monomers selectively cuts graphene into congruent microscopic patterns. These results indicate that the translocation of a large molecule through monolayer CVD graphene can be realized by applying an external driven force to the molecule, and the charge of the molecule has a greater impact on the translocation than the molecule size.2 wafer piece27 and covered by a monolayer of graphene (ca. 1 × 0.5 cm2) from CVD. The typical surface morphology of CVD graphene with micrometer size wrinkles is revealed by atomic force microscopy  mapping results suggest that the whole graphene sheet is rather homogeneous with low defect concentration , the ligand  for polymerization   readily gave poly(methyl methacrylate) (PMMA) brush layers as shown in Fig. −1) Fig. . The for−1) Fig. . Interes−1) Fig. , which a∗1) and without (∗2) initiator-SAM/polymer brush underneath, as confirmed by AFM  on graphene due to the translocation of MMA. Firstly, the ID/IG intensity ratio  increased from ca. 0.09 to ca. 0.22  was lifted from the substrate by the grown PMMA brush underneath.The graphene coverage was fully retained at both regions with   of graphene at this region (∗1) was unchanged   was converted as described above. Again, the SI-CRP of SPMA resulted in PSPMA brushes but apparently the graphene was cut into hexagons congruent to the initiator-SAM pattern Fig. , 17. Ram∗2, Fig. . Raman s∗1) Fig. . And theged Fig. , suggest−1 (without graphene) to 100 nm h−1 ethyl trimethylammonium chloride, METAC) was investigated. As for the anionic monomer, also the transport of METAC through the graphene was significantly slower as revealed by the slower polymer brush growth rate (decreased by 60% from 266 nm hSimilar translocation behavior was observed for another cationic monomer (4-vinylpyridine (4VP), Supplementary Fig. 35, to study the kinetic energy change ΔEK during the translocation process through the graphene nanopore of various edge terminations for the three monomers SPMA, MMA, and METAC  is found for the negatively charged SPMA  has been found for neutral MMA. To quantify the degree of distortion of the nanopore during the translocation process, the change in the pore size (DH) was calculated by computing the distance between diametrically opposed OH groups in the functionalized pore at each simulation time step, and then performing an average over the calculated distances , followed by METAC (DH ≈ 7.86–8.04 Å) and SPMA (DH ≈ 7.92–8.24 Å). In the case of ether termination10, both quantities, τ and ΔEK, smoothly decrease upon increasing the ether concentration, since the hole diameter gets larger and, hence, the monomers have less interaction with graphene defect edge to pass through  approachsee Fig. , 25. We ΔEK Fig. . In addiΔEK Fig. . For theThe combined experimental and simulation results indicate that electrostatic interactions between charged species (monomers) and the functionalized graphene nanodefects are dominating the translocation process. Moreover, the much higher energy loss experienced by the charged monomers may explain (i) the strong obstruction effect observed in the translocation of both cationic and anionic monomers, and (ii) the observed chemical cutting (in the case of anionic monomers), since the larger dissipated energy into graphene may lead to a larger degree of structural distortion of the nanodefects.38, which either enlarges the defects or completely breaks graphene depending on the charges of monomers. (ii) After vertical translocation, the monomers are able to transfer laterally underneath of graphene lattice, which accumulates as polymer brush under graphene and gradually delaminates graphene from substrate24. Both of these two processes are ensured by the atomic thinness and flexibility of graphene monolayer21 and strong Coulomb interactions between monomer and surface-bonded initiator in SI-CRP.We suggest that there are two possible passways for monomers transport at the graphene interface during SI-CRP Fig. : (i) Ver39 of two-dimensional materials into desired patterns or shapes for heterostructures40.In conclusion, we found that large molecules such as vinyl monomers of different sizes can pass through single-layer CVD graphene if driven by a proximal chemical reaction such as SI-CRP. Furthermore, the SI-CRP continues and the formation of polymer brushes indicate a constant flow of monomers through the graphene. Notably, we found that the transport process is more charge-selective than size-selective. For example, a neutral monomer could smoothly translocate to the other side of graphene, by enlarging the graphene native defects. However, the translocation of cationic and anionic monomers was significantly obstructed (60 and 65% in polymerization rates), due to the strong interactions between charged monomers and graphene defects. These results on the translocation of actual molecules through a single layer of a two-dimensional material are intended to trigger further studies on the transport phenomena through this exciting class of materials. An additional aspect of this work is the possibility of the chemical cutting with techniques used for nano- and micropatterned polymer brushestBuMA, OEGMA475, and 4VP were purchased from Sigma-Aldrich , purified before use by passing through a basic alumina column to remove the inhibitor. 3-SPMA, METAC, 2-BiBB, 3-aminopropyltriethoxysilane (APTES), PMDETA (99%), dichloromethane , DMSO, triethylamine (TEA), acetone (dry), and methanol  were used as received. For reactions and water contact angle measurements, bidistilled deionized water was used. Cu plate : 1-side polished, p-type (boron), total-thickness-variation (TTV) < 10 µm, 1–10 Ohm cm; 10 nm Ti adhesion layer; 200 nm Cu (purity >99.9%), root-mean-square roughness <10 nm. The copper plate was consecutively washed with portions of 3 M HCl (in methanol), methanol and ethanol under ultrasonication (2 min), and dried under a flow of argon. The cleaned copper plate was immediately used for reactions.MMA, 2O2:H2SO4, 1:3 v/v, 90 °C, 45 min; WARNING: Piranha solution reacts violently with organic matter!), washed extensively with bidistilled water, and dried with a stream of dry argon.Silicon wafer pieces with a 300 nm oxide layer were obtained from Wacker AG, Burghausen, Germany. To remove any grease or other contaminants, silicon substrates were cleaned with piranha solution  APTES solution in dry acetone and subjected to treatment with ultrasound for 45 min. After SAM formation, the samples were extensively rinsed with dry acetone and dried under argon atmosphere. The substrate was then immersed in dry DCM (20 mL) under a nitrogen atmosphere. TEA (0.4 mL) was added, followed by dropwise adding initiator BiBB  at 0 °C, and the reaction solution was allowed to stand for 24 h with stirring at 20 °C. The substrate was removed, washed with DCM, water, ethanol, and acetone, and then dried under a nitrogen stream. The quality of this surface-bonded initiator preparation method was confirmed by X-ray photoelectron spectroscopy (XPS) and water contact angle measurements. The total surface layer thickness  was ca. 2.0 nm, as judged by ellipsometry.2 by UV illumination (200 W Hg (Xe) lamps, LOT-oriel, Germany) through a photomask. The samples were clamped with Cu TEM grids and irradiated for 45 min at a distance of ca. 10 cm.Patterned initiator was achieved on a selectively etching of as-prepared uniform initiator on SiO2, one side of the single-layer graphene (graphene supermarket) was coated with PMMA resist  and cured at 90 °C for 10 min. The other side of the sample was exposed to O2 plasma to remove the graphene on that side. The Cu substrate was etched away by an aqueous solution of ammonium persulfate (0.25 g mL−1) over a period of 2 h. After being rinsed thoroughly with deionized water, the PMMA-graphene film was transferred to a target substrate. The samples were naturally dried in air for 1 h and stored in high vacuum at room temperature for 24 h to enhance the adhesion of graphene with BiBB-SiO2 surface. PMMA was removed by thorough rinsing in acetone and cured in isopropyl alcohol.To transfer graphene to the initiator (BiBB) modified SiO2O and 0.5 mL methanol, 18.4 μL PMDETA; (iii) SPMA: 1 g monomer, 1 mL H2O and 0.5 mL methanol, 18.4 μL PMDETA.Polymerization solution preparation: (i) MMA: 1 mL monomer, 0.5 mL DMSO, 18.4 μL PMDETA; (ii) METAC: 1 mL monomer, 1 mL H2 was sandwiched with a copper plate  at a distance of D = 0.5 mm adjusted by two spacers29. A drop (ca. 20 µL) of monomer solution prepared above was introduced to the confined space between copper plate and graphene-BiBB-SiO2. The assembly was left for typically 1 h at room temperature. After reaction, the plate was separated and the substrate immediately washed with either fresh DMSO or methanol and water mixture. Finally, the substrates were exhaustively rinsed with acetone to remove all traces of monomer solution, and subsequently dried in a stream of nitrogen.The graphene-BiBB-SiO41 for the C, H, O, N, and S atoms. In all simulations, explicit water has been considered as parameterized in DFTB+ code, in which the interactions among water molecules are given by dipole–dipole as well as van der Waals interaction. In order to take into account van der Waals forces, dispersion corrections were included via Lennard–Jones potentials. The geometry optimization for the monomers and edge-terminated graphene was performed until the absolute value of the inter-atomic force lies below 10−4 atomic units. Periodic boundary conditions in all supercell directions were applied. We first generated a graphene pore defect with CH3 termination and, then, the influence of OH termination concentration is studied. The influence of ether termination on the monomers translocation process through an initially OH-passivated pore has also been analyzed . The Si peak at 520 cm−1 was used as a reference for wavenumber calibration. The peaks were fitted with a single Lorentzian line shape to determine peak position and full width at half maximum. The optical microscopy images were measured on Zeiss Axioskop 2 MAT microscope.AFM scans were recorded with an Ntegra Aura (NT-MDT) atomic force microscope with a SMENA head in the semicontact mode. The used probes have a typical curvature radius of 6 nm, a resonant frequency of 47–150 kHz, and a force constant of 0.35–6.10 N mSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3


Proliferation of retinal pigment epithelium (RPE) is typically observed in limited ocular disorders, in connection with the local mechanism of RPE proliferation-mediated wound repair. Bilateral and diffuse type RPE proliferation is considered to be associated with paraneoplastic syndromes, such as a bilateral diffuse uveal melanocytic proliferation. However, other reported diseases that induce bilateral diffuse RPE proliferation are quite rare, especially for patients who are considered to have a non-malignant status.The bilateral eyes of a 47-year-old woman with bilateral ocular inflammation, presented united multiple small to medium white retinal lesions during the disease progress.Optical coherence tomography showed scattered serous retinal detachments, choroidal folds, choroidal thickening and diffuse RPE proliferation. As autofluorescence and angiography showed a “giraffe pattern”, bilateral diffuse uveal melanocytic proliferation was suspected. However, systemic investigations identified no malignancy. In consideration of the above findings, choroidal inflammation was thought to be the major cause of this condition.The patient was administered intensive systemic steroids. Over the next 2 months, the amount of steroid was tapered off.After administration, the bilateral diffuse RPE proliferation settled down. During the 2-year follow-up, there was no recurrence of ocular inflammation and diffuse RPE proliferation, or any other malignancy found.This finding demonstrates that bilateral diffuse RPE proliferation can be generated as a secondary phenomenon of choroidal inflammation in patients with a non-malignant status. BDUMP is a form of a paraneoplastic syndrome that causes proliferation of the melanocytes bilaterally, which causes visual impairment due to the rapidly progressing exudative retinal detachment and cataract formation. Although it has been reported that the proliferation of melanocytes in the choroid and RPE is considered to be caused by humoral factors produced from tumor, the details of the pathogenesis, pathophysiology and treatment method are still unclear. Other reported diseases that induce bilateral diffuse RPE proliferation are quite rare, especially for patients who are considered to have a non-malignant status.One of the typical diseases that has been reported to cause bilateral diffuse proliferation of RPE is bilateral diffuse uveal melanocytic proliferation (BDUMP).Here, we report bilateral diffuse RPE proliferation, which was a secondary phenomenon caused by an ocular inflammation that was predominantly found to be choroidal inflammation, in a patient considered to have a non-malignant status.2A 47-year-old woman visited an ophthalmology clinic in January 2017 with foggy vision and vision loss. Although her medical history included uveitis 15 years prior, she had no other ocular disease, systemic disease such as inflammatory diseases, or malignant disorders. At the first visit, the best corrected visual acuity (BCVA) was 20/30 and 20/40 in her right and left eye, respectively, while the intraocular pressure was 13 and 11 mmHg in her right and left eye, respectively. In the anterior segment findings, no keratic precipitate, 1+ cells and 1+ flare in the anterior chamber, and remarkable posterior synechia were detected in both eyes  followed by an oral administration (starting at 50 mg / day) led to improvement in the choroidal folds and choroidal thickening. However, the serous retinal detachment tended to remain. Two weeks after the onset of the disease, bilateral diffuse RPE proliferation was clearly observed. A fundus photo showed multiple, coalescing, circular depigmented plaques and pigmentation, which were considered to be caused by hyperplasia of the RPE Fig. A and B. Based on these findings, we considered BDUMP to be the most probable differential diagnosis of the disease that was associated with the hyperplasia of the RPE. As a result, we performed whole-body investigations using computed tomography, magnetic resonance imaging, positron emission tomography, and echo in order to try and detect the presence of malignant tumors in this patient. However, findings of these evaluations did not suggest the presence of any malignant tumors.During the 2-month steroid treatment, there was improvement of the choroidal folds, choroidal thickening and serous retinal detachment observed in the patient Fig. A–C. Corr3BDUMP is a representative disease that is associated with bilateral and diffuse proliferation of the RPE. The clinical features of this disease include:1.multiple circular or oval orange patches (decolorizing spots) at the level of the RPE,2.multifocal early hyperfluorescence corresponding patches,3.extensive thickening of the uvea with focal nodules,4.exudative retinal detachment, and5.rapid cataract progression.In addition, other typical findings include the recent OCT images of the BDUMP identified hyperplastic images of the RPE and the giraffe pattern observed by angiography and autofluorescence. a systemic investigation was repeated in this case for 2 years from the initial onset. However, there was no malignant tumor ever observed. Thus, the proliferation of RPE in this case was considered to differ from the pathological mechanism of BDUMP.When this case was compared to the typical findings for BDUMP, the common ophthalmological findings included bilateral diffuse hyperplasia of the RPE and serous retinal detachment. Therefore, we listed BDUMP as a differential diagnosis in this case. However, extensive thickening of the uvea with focal nodules and rapid progression of cataract, which are typical features of BDUMP, were not observed in this case. In addition, since BDUMP is considered to be a paraneoplastic syndrome and is often observed prior to a malignant tumor, The specific findings in these cases were the formations of local nevus-like elevated lesions, which suggested the proliferation of choroidal melanocytes. On the other hand, our current case did not exhibit any elevated lesions.In this case, we strongly suspected that inflammation played an important role in the pathogenesis of the proliferation of RPE, as OCT showed that the anterior segment inflammation was clearly observed and there was choroidal folds/choroidal thickening. Furthermore, IA showed that there was a filling delay of the choroidal large blood vessel, thereby indicating choroidal inflammation. Although VKH was considered as a differential diagnosis in this case, diffuse proliferation of the RPE and the presentation of the giraffe pattern in this case are not commonly seen in VKH. Moreover, the specific systemic features of VKH, such as aseptic meningitis and sensorineural hearing loss, were not seen in this patient. Therefore, VKH was excluded as a differential diagnosis. It has been reported in previous studies that pseudotumoral retinal pigment epithelial proliferation occurred with VKH. were all ruled out after ophthalmologic and systemic examinations. In addition, local proliferation of RPE induced diseases such as proliferative (diabetes) retinopathy, age-related macular degeneration, and retinal detachment were also ruled out based on the fundus findings.From the aspect of angiography, other differential diagnoses including idiopathic uveal effusion and blood diseases such as lymphoma/leukemia, which cause “giraffe pattern” changes, However, in our current case, steroid treatment was clearly effective on the choroidal inflammation, with a subsequent reduction of the hyperplasia of the RPE observed after the treatment. This strongly supported the theory that bilateral diffuse RPE proliferation was associated with the choroidal inflammation.From a treatment perspective, it has been shown to be difficult to improve the vision in BDUMP patients when just using steroid therapy.In conclusion, the findings described here support the hypothesis that diffuse RPE proliferation can be induced by ocular inflammation, even in patients without any malignant tumors. Furthermore, choroidal inflammation played a major role in generating bilateral diffuse RPE proliferation.Conceptualization: Koju Kamoi.Data curation: Miyuki Tanaka, Natsuko Nagaoka, Tomoka Ishida, Hiroshi Takase.Formal analysis: Miyuki Tanaka, Koju Kamoi.Investigation: Miyuki Tanaka, Koju Kamoi.Supervision: Koju Kamoi, Kyoko Ohno-Matsui.Writing – original draft: Miyuki Tanaka, Koju Kamoi.Writing – review & editing: Miyuki Tanaka, Koju Kamoi, Natsuko Nagaoka, Tomoka Ishida, Hisako Karube, Hiroshi Takase, Kyoko Ohno-Matsui.


Graphene is susceptible to morphological instabilities such as wrinkles and folds, which result from the imposition of thermo-mechanical stresses upon cooling from high temperatures and/ or under biaxial loading. A particular pattern encountered in CVD graphene is that of mosaic formation. Although it is understood that this pattern results from the severe biaxial compression upon cooling from high temperatures, it has not been possible to create such a complex pattern at room temperature by mechanical loading. Herein, we have managed by means of lateral wrinkling induced by tension and Euler buckling resulting from uniaxial compression upon unloading, to create such patterns in exfoliated graphene. We also show that these patterns can be used as channels for trapping or administering fluids at interstitial space between graphene and its support. This opens a whole dearth of new applications in the area of nano-fluidics but also in photo-electronics and sensor technologies. Owing to its atomic thinness, graphene is susceptible to wrinkles and folds. Here, the authors demonstrate controlled wrinkle formation in graphene under uniaxial tension and show that the obtained mosaic patterns can be used as channels for trapping or administering water and other fluids. Such corrugations are of paramount importance for the potential applications of graphene since they can modify the electronic structure and carrier transport, alter surface properties, create electron/hole puddle and induce pseudomagnetic field in bilayers4. The formation of wrinkles, ripples and crumples has been attributed to several physical causes, such as (a) the thermal vibration of the 2D lattice5, (b) edge instabilities, defects/ dislocations6, (c) the mismatch between the thermal expansion of the 2D material and its host substrate9 (d) the evaporation/removal of solvent and its surface tension10, and (e) the relaxation of pre-strained substrate and its surface potential13. Recently, it has been demonstrated that wrinkles in the form of delaminated folds in supported graphenes can be formed either by uniaxial compression or uniaxial tension beyond a certain critical load, depending on the mode of loading. In the first case, the wrinkling direction is perpendicular to the compression axis whereas in tension, wrinkles of the same pattern originate parallel to the loading direction due to Poisson’s contraction exerted laterally by the underlying polymer substrate14.Graphene and 2D materials exhibit interesting morphologies which have been classified as wrinkles, ripples and/or crumplesHerein we demonstrate that in-situ uniaxial tensile testing combined with Atomic Force Microscopy (AFM) can be employed to highlight the formation of complex morphologies in graphene flakes, such as the creation of mosaic patterns similar to those observed in CVD graphenes upon cooling from high temperatures (~1000 °C).15. Appropriate flakes deposited directly on the PMMA were located using the optical microscope and the exact thickness of each flake was identified by the corresponding Raman line of the 2D spectra.In the present study we used a 1-mm-thick sheet of poly-methylmethacrylate (PMMA) as substrate to simply support monolayer (1LG) and bilayer (2LG) graphene flakes that were prepared by mechanical cleavage from HOPG (High Order Pyrolitic Graphite) with the scotch tape method16 in both the loading and the transverse directions. At rest the flakes appear to be quite flat having only a small and smooth fluctuation of ±0.5 nm in the out-of-plane direction which corresponds to the surface roughness of the polymer substrate, in agreement with measurements conducted previously14  was adjusted under an AFM microscope (Dimension Icon Bruker), which allowed the visualization of the flake morphology under incremental uniaxial strain levels (in steps of 0.1%). Very importantly, a dual threaded leadscrew drives the jaws symmetrically in opposite directions, keeping the specimen centred in the field of view during the tensile loading. The graphene flakes were subjected incrementally to tensile strain and the AFM images were captured at various levels of loading. AFM images were acquired with a Scanasyst-air probe of stiffness ~0.40 N/m and the measurements of the topography of the flakes were selected by using the operating Peak Force Nano-mechanical mode. The critical strain for the out-of-plane wrinkling and the wrinkling pattern development beyond a critical strain has been experimentally observed and quantified by using the proposed methodology. The experimental findings for the flakes under pure uniaxial tension are presented in Fig. 00 was adved Fig. . As noteIt is interesting to note that, upon unloading, the lateral wrinkles previously created upon tension are still present since they have been detached from the substrate and due to the interface slippage, the system does not fully recover. In addition, the formation of new wrinkles now perpendicular to the loading direction is observed Fig. . The coe21 and has also been simulated by computational modelling21. It constitutes a unique form of failure initiation which is the main characteristic of compression  of such systems. On the contrary flakes fully embedded in a matrix have been found to exhibit the expected Eulerian sinusoidal mode of compression failure22 due to the presence of constraints for fold creation by the surrounding matrix.The formed wrinkles are in fact isolated folds which have been detached from the underlying substrate and control tests reveal that do not come from any undulations of the surface of the substrate  the graphene flake will be subjected to compressive strain, which can be of sufficient magnitude to induce longitudinal wrinkling, if exceeds the critical strain for wrinkling The threshold of fold formation in the lateral direction seems to be accompanied by interfacial failure or slippage of the whole flake giving rise to the formation of folds in the longitudinal direction Fig.  upon unlsee Fig. . Hence, 21; hence, it is possible to give an estimation of the minimum strain to apply to the substrate in order to create the mosaic morphology for the system at hand, which is approximately 1.8%. The proposed mechanism has been verified by independent mechanical experiments combined with Raman spectroscopy  stresses induced due to the mismatch of the thermal expansion coefficients between copper and graphene result in a compressive biaxial strain of 2–3% in the graphene at room temperature. Such residual strain is far higher than the critical strain required for graphene wrinkling (buckling) thus giving rise to a network of large out-of-plane wrinkles or folds8. It is interesting to note that the peripheral equi-biaxial strain creates circular mosaic morphologies in contrast to the square patterns obtained here. In our case, the final lateral size in the mosaic morphology is essentially determined by the density of wrinkles developed during the loading and the unloading stages. The higher dimensions of the domains developed in the 2LG flake is compatible to a lower wrinkles density, compared to 1LG, in agreement with theoretical predictions14. These features are strongly dependent on levels of adhesion between graphene and the underlying polymer; hence, the use of different substrates can be thus exploited to tailor the final morphology of the mosaic.The mechanically self-assembled mosaic pattern formed here is very similar to the island-like morphology of CVD graphene grown on a copper substrate24. These typical features of the mosaic could be of paramount importance since it may broaden graphene application in the areas of electronics, photonics, nano-fluidics and bio-engineering4. For instance, the strong coupling between localized deformation and the electronic structure that is expected at the junctions could be exploited by strain engineering. Furthermore, constructive use of the self-assembled wrinkle networks could be harnessed as surface texturing agent to direct cell alignment and morphology in tissue engineering25 or for the creation of nano-channels in nano-fluidic devices transporting liquids. In light of possible application, the stability of the wrinkles to cyclic loading, which is particularly important for the off/on controlled applications of the mosaic, has been also verified  for several days. The selection of bilayer mosaic for this feasibility study has been encouraged by recent results of a molecular dynamics study of pressure-driven water transport through graphene bilayers which suggests that, as the channel width increases, the water flow rate increases monotonically and also the increase of channel thickness leads to weaker water-graphene interaction and thus higher water molecule mobility29. The AFM images acquired immediately after the exposure reveal that triangular and polygonal blisters nucleate at most junctions  is a photoresist polymer and was spin coated on the top surface of a PMMA film at ∼2000 rpm creating a layer of ∼300 nm thickness. The samples were characterized by means of AFM (see below) and Raman spectroscopy, by InVia (Renishaw) microspectrometer with 2400 grooves/mm grating for the 785 nm laser excitation; Raman spectra were measured at and the laser power was kept at 1.2 mW on the sample to avoid laser-induced local heating. A ×100 objective with numerical aperture of 0.95 was used in all cases. Experimental data were fitted by using Lorentzian functions, one for the monolayer and four for the bilayer graphene.The tensile experiments were conducted using a micro-tensile tester (Deben MT 200) that could be adjusted under AFM for simultaneously applying strain and acquiring images under AFM. The increment step of strain was 0.1% for all cases. The AFM images were obtained using Dimension Icon microscope (Bruker) operating in Peak Force Tapping mode using ScanAsyst-Air probes . No data treatment apart from line subtraction (retrace) to remove the tilt has been performed. Wrinkle amplitudes and half wavelengths were measured from line profiles manually for each wrinkle. Medians of the thus obtained parameters were then used to characterize the wrinkles in each profile. Also, lateral size of the islands delimited by the wrinkles has been estimated by averaging the length of two crossed lines across the island measured manually from line profiles.Tensile experiments have been performed using the same set-up under the Raman microscope (InVia Renishaw microspectrometer) by using the 785 nm laser excitation. At each deformation step, several Raman spectra were acquired in different location of the 1LG flake under investigation (near the geometric centre) and experimental data were fitted by using one Lorentzian function.32.High humidity exposure experiments were performed at 90% RH and at RT in a custom-made climate chamberSupplementary Information


The new immunotherapy targeting the programmed cell death 1 (PD-1) receptor and its cognate ligand PD-L1 has renewed hopes of eradicating the most difficult human cancers to treat. Among these, there are the poorly differentiated and anaplastic thyroid cancers, unresponsive to all the therapies currently in use. In the present review we will summarize information regarding the expression of PD-L1 in the different thyroid cancer histotypes, its correlation with clinicopathological features, and its potential prognostic value. Then, we will evaluate the available data indicating the PD-1/PD-L1 axis as a promising target for thyroid cancer therapy.  BRAF and RAS oncogenes, and chromosomal translocations of the RET (REarranged during Transformation) and NTRK1 (Neurotrophic Tyrosine Kinase Receptor 1) genes, which lead to the activation of a common carcinogenic pathway, i.e., the MAPK/ERK signaling . ,63. 8,63 noticed ,37,64,67ild type . These fn (Treg) . From thn (Treg) . These vanalyzed . AltogetV600E and lymph node metastasis, but not with other clinicopathological features or DFI [Regarding PD-L2 expression in thyroid tumors, to the best of our knowledge, only two studies have been performed so far . One reps or DFI . The sams or DFI . In any Over the last decade, the considerable increase of our knowledge about mechanisms underlying the ability of cancer cells to elude the detrimental action of the immune system has led to a renewed interest for immune-based therapy in oncology, especially for hard-to-treat cancers, including the poorly differentiated and frankly anaplastic thyroid cancers ,81. In pPD-L1 and CTLA-4 genes, the canonical BRAF-like and RAS-like PTC were classified into 2 subgroups each: BRAF-IR (immunoreactive) and RAS-IR, characterized by up-regulation of immune-related genes and tumor infiltration by several immune cell subtypes, including T cells; and BRAF-ID (immunodeficient) and RAS-ID, characterized by low expression of immune-related genes and low infiltration of immune cells [The objective response rate to PD-1/PD-L1 directed therapies varies from 13 to 43% in different tumor types . The besne cells ,87. ConfV600E mutation and PD-L1 protein by IHC in tumor tissue, the patient was treated with vemurafenib (BRAF inhibitor) and nivolumab. Whereupon, the patient experienced a continued reduction of the metastatic lesions with complete radiographic and clinical remission of the disease 20 months after the beginning of nivolumab therapy [At present, no information from clinical trials with anti-PD-1/PD-L1 directed therapies enrolling patients affected by aggressive thyroid cancers is available. However, different preclinical studies demonstrated the efficacy of PD-1/PD-L1 axis blockade in restraining growth of ATC-derived cell lines injected in mice ,88,89. I therapy . In anot therapy . Of thes therapy . Very re therapy . Twenty- therapy . A parti therapy . At the  therapy . This isSince the activation of the MAPK signaling in thyroid cancer has been shown to negatively modulate the immune response in the tumor microenvironment, it is likely that targeted therapies directed against this pathway may revert the immune suppression due to abnormal kinase activation . For exaThe information so far available suggests that PD-L1 could represent a useful prognostic marker for risk stratification of thyroid cancer patients, and that anti-PD-1/PD-L1 directed therapies could be a valid option for patients affected by the most aggressive thyroid cancers, such as PDTC and ATC, unresponsive to the currently available therapies. Some issues, however, still remain to be addressed. In particular, the standardization of the IHC techniques, the interpretation of PD-L1 immunoreactivity in cancer tissues, and a more reliable characterization of biomarkers capable of predicting patients’ response to anti PD-1/PD-L1 therapies.


Chitosan (CHT) is a polysaccharide with multiple claimed properties and outstanding biocompatibility, generally attributed to the presence of protonable amino groups rendering a cationic natural polymer. However, the effect of changes in CHT structure due to hydration is not considered in its performance. This study compares the effects on biocompatibility after drying at 25 °C and 150 °C scaffolds of chitosan, polyethylene glycol diglycidyl ether (PEGDE) crosslinked CHT  and glutaraldehyde (GA) crosslinked CHT. PEGDE crosslinked CHT showed a reduction in free amino groups and the amide I/II ratio, which exhaustive drying reduced further. In X-ray diffraction (DRX) analysis, PEGDE crosslinked CHT showed multiple peaks, whereas the crystallinity percentage was reduced with an increase in PEGDE concentration and thermal treatments at 150 °C. In a direct contact cell assay, high osteoblast viability was achieved at low and medium PEDGE concentrations, which was improved when the crosslinked scaffolds were thermally treated at 150 °C. This was attributed to its partial hydrophilicity, low crystallinity and low surface roughness; this in spite of the small reduction in the amount of free amino groups on the surface induced during drying at 150 °C. Furthermore, PEGDE crosslinked CHT scaffolds showed strong vinculin and integrin 1β expression, which render them suitable for bone contact applications. Tissue repair makes use of different strategies, including scaffold-free, cell-free and traditional tissue engineering, where cells are seeded on biodegradable scaffolds in the presence of bioactive molecules while being stimulated in a bioreactor. Therefore, scaffolds are an important element for tissue repair, because they not only provide mechanical support, but also facilitate cell proliferation and differentiation through their porous structure. The materials used to produce scaffolds are natural or synthetic polymers, ceramics and even biodegradable metals . Among them, natural polymers have caught the attention of several researchers due to their diverse and sometimes unexpected properties, such as reactivity, strength and toughness, when blended with other polymers ,3.Chitosan (CHT) is the N-deacetylated derivative of chitin with a typical DA of less than 0.35. It is thus a copolymer composed of glucosamine and N-acetylglucosamine. The physical properties of CS depend on several parameters such as the molecular weight , DD (in the range of 50–95%), sequence of the amino and the acetamido groups and the purity of the product .CHT has multiple claimed properties, such as biocompatibility ,6,7, antThe content of amino groups, degree of deacetylation and molecular weight are the main reasons for the diverse physicochemical properties of chitosan . In termIn addition, it is known that CHT is a semicrystalline material with a polymorphic crystalline structure that can be transformed from its hydrated forms to an anhydrous or “annealed” form. This quality of the CHT was reported for the first time by Sakurai . Surface properties are determinants of CHT cellular response ,29 but iIn this study, we argued that not only the presence of amino groups on CHT are responsible for its biocompatibility, but also that the existence of other factors, such as changes in crystallinity and surface roughness, played a significant role in its cellular response. Therefore, we proposed the use of polyethylene glycol diglycidyl ether (PEGDE) as a crosslinking agent in order to improve CHT mechanical properties, and to assess their biocompatibility with osteoblasts due to amino groups involvement in the crosslinking reaction. Furthermore, we assessed the effect of a high temperature drying treatment on PEGDE crosslinked CHT to fully appreciate if these structural changes will jeopardize CHT biocompatibility. −1, , while J.T. Baker provided acetic acid . MTS CellTiter 96® Aqueous Non-Radioactive Cell Proliferation obtained from Promega  Dulbecco’s modified Eagle’s medium (DMEM) from Biowest , human osteoblast hFOB1.19 from ATCC® CRL-11372™ . Integrin β1 (A-4): sc-374429, vinculin (7F9) sc-73614 and antibody  were purchased from Santa Cruz Biotechnology .Chitosan (CHT) with viscosity molecular weight of 223,332 gmolmentally ) and a dTg, which depended on water content and heat treatment,  was mixed with 32 μL PMS (phenazine methosulfate) after thawing. The entire procedure was performed at 25 °C and protected from light. The MTS solutions was used immediately after preparation.Cell viability studies were done using CellTiter 963 cells/scaffolds in a 96-well plate. The seeded cells were incubated at 37 °C, 5% CO2 and 95% humidity for 48 h. For the direct contact assays were used samples of 10 mg of weight. Scaffolds were sterilized by UV for 15 min. Osteoblasts, obtained from human osteoblast ATCC hFOB1.19, were seeded at 2 × 10® syringe filter of 0.22 μm. For an indirect contact test, extracts replaced the culture medium in a 96 well plate with 2 × 103 cells/well. The seeded cells were incubated at 37 °C, 5% CO2 and 95% humidity for 48 h.Extracts were obtained according to the practical guide for the preparation of samples and reference materials, ISO 10993-12 . 25 mg oFor both techniques (direct and indirect) a positive control was included which comprised cells with only culture medium (C+), with a negative control (C−), with hydrogen peroxide and with the target with MTS solution, as well.® was added to each well. This was incubated for 3 h and the absorbance was measured in a multi-well spectrophotometer (Thermo Scientific™) at 490 nm.After 48 h, 20 μL of MTS CellTiter 963 cells/mL (osteoblast ATCC hFOB1.19), and incubated at 37 °C, 5% CO2 and 95% humidity for 48 h. The samples were rinsed with PBS, fixed with 4% formalin and blocked with 1% ABS tween. The first antibody, Integrin β1 (A-4): sc-374429 was added (50 µL per plate diluted with PBS-tween 1:150) and incubated at 4 °C overnight. After washing with PBS, the second antibody  and left for 2 h. The samples were observed in a Leica fluorescence microscope equipped with a digital camera, using the LAS AF software . The same procedure was carried out for vinculin, where the antibody (7F9 sc-73614), was used. All antibodies were provided by Santa Cruz Biotechnology .Cytoskeletal alterations were measured by the expression of integrin and vinculin adhesion proteins. To carry out these tests, extracts obtained from chitosan scaffolds crosslinked with PEGDE were used. A 24-well plate with 120 µL of each sample was seeded with 20 x 10−1 and 3000 cm−1 (peaks at 3360 cm−1 and 3285 cm−1) corresponding to the O–H (from chitosan and water) and vibration of N–H in N–H2 groups. At 2920 cm−1 and 2850 cm−1 absorptions corresponding to stretching C–H in C–H2  were observed. While the absorption band at 1650 cm−1 was associated to C=O vibration in the amide I N–H (acetamide) group, the band at 1560 cm−1 was attributed to C–N and N–H2 bending in amide II region. At 1422 cm−1 bands related to CH2 bending were observed. FTIR spectra of chitosan films dried at 25 °C and at 150 °C are shown in −1 an absorption which can be related to CH2 wagging appeared; and finally, at 1319 cm−1, the amide III appeared. Amide III is commonly used for calculating the degree of acetylation. Skeletal vibrations characteristic of the chitosan structure appears at 1150 cm−1 (C–O–C), 1080 cm−1 and 1030 cm−1 (pyranose ring). At 1380 cm−1 became composed of the amide I and the corresponding imine bond [−1), however amide II was of higher intensity than amide I absorption. The Fourier-transform infrared spectroscopy (FTIR) spectra of chitosan films crosslinked with PEGDE showed a higher intensity of amide I in comparison with amide II. This difference was even more marked as the amount of PEGDE increased. The amino groups, now at 1577 cm−1, are more reactive than hydroxyl groups, and can react with the epoxy ring leading a crosslinked structure while showing lower intensity at the highest concentration of PEGDE  was reduced significantly, in comparison with the band at 1560 cm−1 (amide II), which shifts on drying. During GA crosslinking, amide II was smaller than amide I, and the absorption at 1380 cm−1 remained unchanged, but at a lower intensity as to the band at 1420 cm−1. In PEGDE-crosslinked CHT, a higher intensity of the amide I was observed again, in comparison with amide II. This indicates that either these bands are not a good reference to estimate the degree of crosslinking, or that the crosslinking mechanism is different for GA rather than for PEGDE crosslinking. However, the broad O–H and N–H bands located at 3360 cm−1 were of higher intensity compared to the vibration of C–H located at 2922 cm−1 in contrast to the observed behavior in CHT alone and GA-crosslinked CHT. Finally, absorptions at 1380 cm−1 and 1315 cm−1 were of higher intensity compared to the band at 1418 cm−1.The infrared spectra of the cross-linked films dried at 150 °C are presented in −1 to 3445 cm−1, and the shifting of the amide I band from 1635 cm−1 to 1655 cm−1. Our study shows that that drying at 150 °C reduces O–H intensity . This outcome shows that interaction of adsorbed water molecules is diminished not only in the 3400–3200 cm−1 region, but also in the carbonyl region, which also implies that dehydration takes place not only at the surface level, but also in the bulk of chitosan. Regarding, the amide I band, there was no apparent shift; nevertheless, it exhibited a significant reduction in intensity. This agrees with Zawadzki et al. , in whicThermal treatment at 150 °C yields an insoluble chitosan scaffold when placed in acetic acid. After thermal treatment at 150 °C it is possible that some chemical reactions involving amino groups lead to the crosslinking of the chitosan scaffold, for example oxidation of alcohols render aldehydes, which in turn reacted with the amino groups, leading to imine formation. This explain why the percentage of the amino groups quantified at 150 °C was very low. These results should be taken with caution, as it is possible that acetic acid was either not completely eliminated, not only from dissolution and film preparation, but also from purification of the raw product during manufacture; or because commercial chitosan employed was in partially protonated form. In addition, Balázs  mentionsThe binding and growth of cells in chitosan substrates has been attributed to the cationic nature of chitosan amine groups. Data suggest that, as the level of deacetylation is raising, the positive charge density of the chitosan increases; consequently, there is an increase in the attraction of negatively-charged cells. Factors such as the degree of deacetylation and chemical modifications influence the hydrophilic properties of chitosan films and their interactions with the biological environment ,41. ThesThermal stability can be evaluated as the onset temperature of the degradation step, the maximum of the decomposition temperature and the temperature corresponding to the 50% weight lost. In this study we use the maximum temperature and the 50% weight lost to compare the thermal stability.It is observed that the previously dried CHT at 150 °C shows a lower weight loss as a result of the drying process that removes part of the adsorbed water. However, the weight loss related to water above 100 °C is due to hydrogen bonds formation between functional groups in CHT molecules and water . In the The greater weight loss for CHT dried at 150 °C, compared to the dried at 25 °C, could be due to the fact that the second stage of chitosan degradation began around 150 °C , so the In relation to CHT crosslinked with PEGDE dried at 25 °C, a decrease in weight loss corresponding to adsorbed water is observed in comparison with pure chitosan. However, at higher PEGDE concentrations, adsorbed water remains the same. This could be associated with crosslinking between chitosan and PEGDE, which would decrease the absorption of water in the material . AdditioLikewise, more water was present in samples dried at 25 °C than in those treated at 150 °C. However, the mass loss in the second stage of degradation was similar. For CHT dried at low temperatures, the amount of retained water was higher in GA crosslinked CHT than in PEGDE crosslinked CHT, demonstrating its higher efficiency as a crosslinker for rendering a low water absorbing hydrogel. For pristine CHT, water is lost in the first stage; whereas, in the second stage, chitosan main chain degradation (depolymerization) and the elimination of volatile products, from pyranose ring destruction and deamination, is expected as reported by Zawadski et al. . NonetheFinally, for all CHT samples crosslinked with PEGDE and dried at 150 °C, it is generally observed that they exhibited better thermal stability than pure chitosan and chitosan crosslinked with PEGDE at 25 °C. This could indicate that the drying treatment at 150 °C promotes a better crosslinking between the chitosan and the PEGDE, which renders samples with higher thermal stability. In other words, CHT-PEGDE dried at 150 °C exhibited a smaller loss of mass (53–54%) at interval of 300–700 °C compared to CHT (72%). In the same way, all samples of CHT-PEGDE dried at 150 °C revealed a smaller loss of mass than CHT-PEGDE at 25 °C (58–70%).Petrova et al. reported the lost solubility of their samples, attributed to the formation of ionic crosslinks upon dehydration ; we obseThis highly ordered structure arises from the hydroxyl and amino groups which can form strong intermolecular and intramolecular hydrogen bonds. This structure was retained with a slight degree of crosslinking (CHT PEGDE1), but giving rise to additional peaks at 2θ = 5.6° and 2θ = 15° in more crosslinked samples (CHT PEGDE2 and CHT PEGDE3). Baroudi et al. attribute the 2θ = 15° to a hydrated chitosan polymorph crystal as a complex with water and acid .When GA was used as the crosslinking agent, the amorphous halo became more evident (peak at 2θ = 19.7°) which indicates the loss of the crystalline structure. Regarding the slightly PEGDE crosslinked CHT, while the peak at 2θ = 20.1° tended to disappear, the peak at 2θ = 9.58° not only reduced but shifted to lower angles—this outcome suggests an increase in chain separation. Interestingly, CHT PEGDE2 showed multiple peaks  denoting a heterogeneous structure; contrary to CHT PEGDE3, a more crosslinked sample, which clearly showed only two reflections at 2θ = 9.68° and 2θ = 20.2°.I) of each of the films. The table shows how crystallinity decreased with the degree of crosslinking and with drying at 150 °C.There are reports that suggested thermal treatments on chitosan can change its physical properties, affecting not only its appearance but also its aqueous solubility and rheology ,51,52. OIt has been reported that the anhydrous chitosan produced through exhaustive dehydration processes has an identical crystal structure to hydrated chitosan. This is, characterized by having an orthorhombic unit cell with a = 7.76 Å, b = 10.91 Å and c = 10.30 Å  as well as a = 4.4 Å, b = 10.0 Å and c = 10.3 Å . However, instead of containing four chains of chitosan with eight water molecules, only two chains were involved .I), as the water can easily penetrate amorphous regions. This was not observed, as Td1 was 15% when the sample was dried at 25 °C, and only 9% when the sample was dried at 150 °C for the pristine chitosan. This can be explained by the used formula and the presence of multiple peaks in the diffractogram. It was expected that more water was present in samples with a lower crystalline index , but only one peak at 399.5 eV was observed, and broadens during its drying at 150 °C.The XPS survey spectra of chitosan films dried at 25 °C and 150 °C are shown in GA crosslinked CHT exhibited three types of carbons at 25 °C, but they tend to disappear with drying at 150 °C, i.e., the C1s band broadens. GA crosslinked CHT showed also a symmetrical O1s peak for films dried at 25 °C and 150 °C. Finally, N1s become broader as thermal treatment increased; however, three peaks  at low temperature drying were observed. This is a clear evidence of the crosslinking reaction.In PEGDE crosslinked samples, three types of C1s were observed, but when PEGDE at medium and high concentration was used, C–O (286.4 eV) were clearer (more at.% contribution) in samples treated at 25 °C; nevertheless, it regained its original intensity when dried at 150 °C. PEGDE crosslinked CHT showed also a symmetrical O1s peak for films dried at 25 °C and 150 °C. For the case of N1s, no visible changes were observed at low concentrations (PEGDE1), at both drying conditions, but N1s became either broader or with two broad peaks (399 eV and 402.5 eV) at the higher concentration (PEGDE 3) when dried at 25 °C. Ra) of the films dried at 25 °C  are shown in Polar groups introduced by different procedures tend to be buried in bulk when they enter in contact with air for a prolonged period; however, they remain on the surface when they are in contact with water or any other polar environment . In our These results agree with oxygen surface composition obtained by EDX, as it was clear that oxygen-containing groups were at the surface rendering a more hydrophilic surface. Zhang et al.  report sIn general, surface wettability is strongly associated, not only with surface contact angle, but also with surface free energy, and it is affected by diverse aspects such as surface elemental composition and roughness. Wettability is perhaps the most important factor to determine the quantity and quality of adsorbed proteins, which in turn affects cell adhesion . Yinghui et al.  noted thChitosan scaffolds must be able to support cell adhesion, and the growth and development of the extracellular matrix, as well. Cell adhesion and proliferation in chitosan substrates has been attributed to the cationic nature of chitosan amine groups .This means that high cell viability in direct contact was achieved in pristine semicrystalline (CrI = 64.1%) CHT dried at 25 °C with 72.5% of free amino groups, Ra = 45.8 ± 4.6 and 72° of DMEM contact angle and in semicrystalline (CrI = 51.3%) PEGDE1 crosslinked CHT dried at 150 °C with 55.3% of free amino groups, Ra = 28.2 ± 7.9 and 57° of DMEM contact angle. This also means that, for a lower amount of amino groups, a higher hydrophilicity renders higher viability in the presence of a less semicrystalline structure. GA crosslinked CHT dried at 25 °C exhibited the lowest viability in direct contact with 51.5% of free amino groups, Ra = 26.3 ± 6.3 and a DMEM contact angle of 56°. Even when the contact angle is lower than pristine CHT, less amino groups are available, resulting in a lower viability. Furthermore, glutaraldehyde is considered as a toxic crosslinking agent that rendered an amorphous (CrI = 21.5%) and smooth CHT surface allowing little cell attachment.The distribution of the cells in scaffolds dried at 25 °C and 150 °C was observed after 48 h of incubation by either direct or indirect contact . A betteJayakumar et al.  evaluateVinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions, and also to cell-biomaterial interactions. The ability to generate and transmit forces depends on the strength of the connection between the integrin adhesion receptors and the actomyosin cytoskeleton mediated by focal adhesion proteins. Vinculin connects the actomyosin cytoskeleton with β integrin subunits through binding to talin, or with the α4 and α9 integrin subunits through binding to paxillin, respectively. In the adhesion test, using the integrin antibody the obtained images are shown in The characteristics of the polymer surface could affect the amounts and types of bound proteins, as well as the conformation, orientation, or binding strength of the adsorbed protein. Zhang et al.  reports Although all PEGDE crosslinked CHT scaffolds support cell adherence, it is worth bearing in mind that that cell adhesion, as demonstrated in this study, is not indicative of how supportive a substrate is to cell spreading, and that cell spreading does not correlate with focal contact formation .The results obtained demonstrate that there are several chemical and structural differences between CHT scaffolds dried at 25 °C and 150 °C, as shown by various techniques. FTIR showed that amide II/amide I ratio changed as the drying temperature was increased in pristine CHT and GA crosslinked CHT, and that the formation of a more amorphous polymer or with reduced crystallinity was observed as demonstrated by XRD. In addition, the decomposition temperature (Td2) tends to increase as demonstrated by TGA. When the scaffolds were crosslinked with PEGDE at medium and high concentrations, a similar behavior was observed, i.e., a reduction in free amino groups, amine I/II ratio and crystallinity. However, osteoblast viability and adhesion was promoted by PEGDE crosslinked CHT at low and medium concentrations, mainly due to its hydrophilicity (low contact angles due to the presence of oxygen containing species), its low crystallinity and its low surface roughness, in spite of the small reduction in the amount of free amino groups on the surface induced during drying at 150 °C. Finally, these results suggest that PEGDE at low concentrations is a suitable biocompatible crosslinker agent for chitosan; and, consequently, these materials are can be used in bone contact applications


The significance of epithelial‐mesenchymal transition (EMT) and immune checkpoint proteins in thymic carcinoma remains unknown. We examined the clinical significance of EMT, tumor‐infiltrating lymphocytes expressing the immune checkpoint protein, programmed cell death 1 (PD‐1 + TILs), and the expression of PD‐1 ligand 1 (PD‐L1) in thymic carcinoma (TC). We also investigated the relationships between these immune checkpoint proteins and the EMT status and examined the impact of induction chemotherapy on patients with tumors that express these proteins.The relationship between PD‐1 + TILs/PD‐L1 and clinicopathological findings including EMT was investigated by immunohistochemistry (IHC) of surgically resected samples from 43 patients with TC. In 15 patients receiving induction therapy (IT), those factors were compared before and after IT.P = 0.0095, PD‐1; P = 0.001, PD‐L1; P = 0.0037). We found a significant relationship between PD‐L1 and EMT status (P = 0.01). In patients who received IT, PD‐L1 increased, and the change was strongly correlated with EMT status (P = 0.01).With IHC, 26 cases (60.5%) were positive for PD‐L1, and 19 cases were positive for PD‐1 + TILs (44.2%). The disease‐free survival rate in patients showing EMT and who were PD‐1/PD‐L1 positive was significantly worse compared to negative cases (EMT; Epithelial‐mesenchymal transition, PD‐L1, and PD‐1 + TILs have prognostic impact, and PD‐L1 is correlated with EMT status. PD‐L1 expression after IT was significantly higher compared to before IT and was correlated with the EMT change. Thus, PD‐L1 may be upregulated during EMT, and anti‐PD‐1/PD‐L1 immunotherapy may provide reliable treatment of TC in combination with chemotherapy. Because of the small number of cases, the biological and oncological characteristics of TC are poorly understood, and no standard treatment strategy has been established. Surgery, chemotherapy, and radiation therapy are performed to treat TC. Surgery is often the first treatment in clinical practice when complete resection appears possible according to preoperative imaging. However, tumor invasion of surrounding tissues and regional lymph node metastasis frequently occur. In patients with advanced clinical stages, multimodal therapy such as induction chemotherapy or chemoradiotherapy (followed by surgery) is performed. Despite those treatments, the recurrence rate and mortality rate remain high.The pathway involving the immune checkpoint proteins, programmed cell death 1 (PD‐1) and PD‐1 ligand 1 (PD‐L1), plays an important role in cancer progression and the cancer microenvironment.Epithelial‐mesenchymal transition (EMT) is a key process in cancer progression and distant metastasis inseveral malignancies including non‐small cell lung cancer (NSCLC).22.1This study was a retrospective analysis of 43 patients with TC whose tumors were completely resected between 1996 and 2017 at Osaka University Hospital . The Institutional Review Board approved this study, and written informed consent for the study and surgery was obtained from each patient. A computed tomography (CT) scan was routinely performed every 6 months as follow‐up.2.2Using clinical samples obtained from enrolled patients with TC, we performed IHC with several antibodies that recognize cancer‐associated proteins, including c‐kit (TC marker), PD‐1/PD‐L1 (immune checkpoint proteins), and E‐cad, N‐cad, and TGF‐β (EMT‐associated proteins). The primary antibodies used in this study are listed in the Data Immunohistochemistry staining for EMT evaluations was performed as in previous reports.PD‐L1 expression was evaluated as the percentage of cells staining positive for PD‐L1 , and PD‐L1 positive was defined as TPS ≥50%. In addition, we defined cases with an increase in PD‐L1 TPS after IT as PD‐L1 change‐positive by comparing PD‐L1 IHC results before and after IT. PD‐1 IHC was evaluated as the extent of TILs after hematoxylin‐eosin staining and PD‐1‐positive TILs (PD‐1 + TILs) using a visually estimated four‐point scale: 0 (absent), 1 (<30%), 2 (30%‐60%), and 3 (>60%). Samples with a score of 0 or 1 were considered negative, and those with a score of 2 or 3 were considered positive.2.3The correlations between clinicopathological factors and variables  were evaluated by Fisher's exact test. Categorical variables including sex, Masaoka stage, and histopathology and continuous variables including tumor size were evaluated with analysis of variance (ANOVA). The histological response to induction therapy was classified into four categories as follows: Ef0: no histological response, Ef1: more than one‐third of the tumor cells viable, Ef2: less than one‐third of the tumor cells viable, Ef3: no viable tumor cells according to General Rule for Clinical and Pathological Record of Lung Cancer.P values <0.05 were considered to be statistically significant.Disease‐free survival (DFS) and overall survival (OS) were analyzed with the Kaplan‐Meier method, and the log‐rank test was used to compare the survival distributions of subgroups. DFS was defined as the time from the data of surgery to the first event of either disease recurrence or death due to any cause. OS was defined as the time from the date of surgery to death due to any cause. The correlation between PD‐L1 IHC status (TPS) and variance was analyzed with ANOVA. The univariate Cox proportional hazards regression model was used to analyze DFS. All statistical analyses were performed with JMP version 13 for Windows . 33.1The median follow‐up time was 4.3 years : 3.0‐5.5 years). Patient characteristics are listed in Table 3.2P = 0.0037, and Figure P = 0.02). In addition, Kaplan‐Meier analysis showed that PD‐L1‐positive TPS patients had a significantly worse DFS rate compared to PD‐L1‐negative patients . A significant relationship between PD‐L1 expression and OS was also found . The relationships between PD‐L1 expression and clinicopathological factors are shown in Table We analyzed the clinical implication of PD‐L1 expression in TC. Figure P = 0.0056). We found no significant relationship for OS regarding PD‐1 expression by TILs . Univariate Cox proportional hazards regression models for estimating DFS showed that the presence of PD‐1 + TILs was one of the prognostic factors . No significant relationship was found between EMT‐positive and EMT‐negative patients for OS . Univariate analyses of DFS also showed that positive EMT status was a prognostic factor (Table P < 0.01). These results suggest that EMT is a potential prognostic factor for recurrence after surgical resection of TC.The clinical significance of the EMT status in TC remains unclear. Here, we investigated the clinical implication of EMT as a prognostic factor in TC and examined the relationship between EMT and clinical backgrounds including PD‐L1 expression with IHC staining of TC specimens. The relationships between the presence of EMT status and the clinicopathological background are shown in Table P = 0.002).Figure 3.4Next, to explore whether these relationships between PD‐L1 expression and EMT status after IT in TC are similar to our previous report of NSCLC,P = 0.004). We also evaluated IHC staining of PD‐L1 and EMT markers including TGF‐β before and after IT in the same patients and compared their status before and after IT. Upregulation of PD‐L1 TPS  showed a significant correlation with EMT positivity and with a change in TGF‐β . In addition, we found a significant relationship between the positive rate of EMT and the TGF‐β change .The PD‐L1 TPS of patients who underwent IT was significantly higher compared with the TPS of patients who did not undergo IT Figure A. Next, 3.5P = 0.03). Regarding OS, we found no significant relationship between the presence or absence of a change in PD‐L1 .Next, we examined the prognostic impact of the presence of a change in PD‐L1 TPS in 15 patients who underwent IT followed by surgery. Figure 4We analyzed IHC staining of EMT markers and the immune checkpoint proteins, PD‐1 and PD‐L1, in clinical samples of TC obtained by surgical resection. Our data suggest that the EMT status and immune checkpoint proteins have prognostic impact and that their expression is closely related. To our knowledge, our study analyzed one of the largest number of TC surgical samples regarding EMT status and PD‐1/PD‐L1 expression.To overcome advanced TC, surgery and chemotherapy are the maintherapeutic options. Despite those multimodal therapies, the outcome is far from satisfactory, and no standard therapy has been established. One reason is that biological research about TC has not made much progress compared to other carcinomas. Because TC is rare, our clinical experience has been insufficient, and oncological characteristics are poorly understood. In the present study, we revealed the microenvironment of TC from the viewpoint of cancer immunology. Recently, immunotherapy has been developed as another option for several malignancies. Agents that block PD‐1/PD‐L1 have exhibited dramatic antitumor efficacy in clinical trials for patients with a variety of cancer types.However, the clinical significance of PD‐L1 in TC is poorly understood. Although a few groups have discussed the clinical significance of PD‐1/PD‐L1 expression in TC with IHC analysis, the significance as a predictive factor is controversial. Katura et al showed that high PD‐L1 expression is correlated with unfavorable prognosis,Epithelial‐mesenchymal transition plays a crucial role in cancer progression, and this phenomenon can be found in the cancer microenvironment in several types of cancers. EMT is promoted by TGF‐β secreted from not only the cancer cell itself but also from other cell types. EMT confers invasive and metastatic abilities on cancer cells that are necessary for metastasis.We hypothesized that PD‐L1, N‐cad and TGF‐β expression was also upregulated after chemotherapy induction in TC. To test this hypothesis, we performed IHC for PD‐L1 and EMT markers including TGF‐β and compared the results before and after IT. Similar to our previous reports regarding NSCLC,In general use of immune‐check point inhibitor (ICI), PD‐L1 IHC assay is used and the results are biomarker for patient selection and therapeutic response of immune‐check point inhibitor (ICI). However, there are several problems such as sensitivity and appropriate cutoff in the IHC assays. From our results, EMT markers may be useful surrogate markers for case selection of ICI after anticancer agent.Our study has some limitations. The first limitation is the small sample size. Therefore, the predictive role of PD‐L1 expression is still controversial. Future studies should involve increased numbers of cases in collaboration with multiple institutions. As a second limitation, our IHC data provide supporting evidence that PD‐L1 expression is enhanced thorough the EMT process or TGF‐β signaling in TC. The mechanism of regulation of PD‐L1 expression needs to be elucidated by utilizing molecular biological techniques, although there are currently no commercially available TC cell lines.In conclusion, understanding the mechanism of PD‐L1 expression will yield important information regarding the role of immune checkpoint proteins in TC. Our results suggest that EMT markers may be informative for making decisions about induction of immunotherapy. In addition, our results suggest that the EMT status plays an important role in cancer progression and has a significant relationship with PD‐L1 expression. Our results also show that PD‐1/PD‐L1 blockade after chemotherapy or in combination with chemotherapy could be more effective than monotherapy and suggest another therapeutic option for treating chemo‐resistant cancer.The authors have declared no conflicts of interest. We state that the material has not been previously published or submitted elsewhere for publication. All authors have read and approved the manuscript and made a sufficient contribution to the work. We will transfer copyright to the Publisher. Click here for additional data file.


Pyrexia of unknown origin (PUO) is a common problem in day-to-day practice. FDG PET CT is an established investigation that aids in identifying the cause of PUO. Due to its high sensitivity PET detects an occult hypermetabolic focus in the body where CT helps in anatomical localization, vascularity, enhancement characteristics of the lesion detected on PET. It helps to differentiate benign versus malignant cause and target biopsy. Tuberculosis, lymphoma, pyelonephritis, thyroiditis appear hypermetabolic on FDG PET CT. Pericardial sarcoidosis is rare and not reported in literature as a cause of PUO. Presented here is a case of PUO secondary to pericardial granulomatosis diagnosed on PET CT. Cardiac MRI also helps in better tissue characterization and associated myocardial involvement of sarcoidosis. Histology confirmed the diagnosis of pericardial sarcoidosis in this case. With reducing cost, PET CT is being increasingly used in cases of fever of unknown origin. Common causes of PUO are bacterial infection as tuberculosis, hidden malignancies as lymphoma, autoimmune diseases as thyroiditis etc. High sensitivity of FDG PET enables early detection of lesions before morphologic changes set in. Other conventional imaging methods largely give anatomical information and depend upon manifestation of morphologic changes. FDG-PET CT is performed as a whole body procedure hence detects number and site of lesions not suspected clinically. We report a case of pericardial sarcoidosis suspected on PET CT and confirmed on histology. A 44 years old male presented with 4 weeks of fever, breathlessness. There was no weight loss (90 kg). Physical examination showed tachycardia 125 beats per minute, tachypnoea (36/minute), normal blood pressure (110/80 mmHg). Soft systolic murmur was heard in left parasternal space. 18F- fluorodeoxyglucose on 6 hours empty stomach. Scanning was done at 60 minutes using Siemens Horizon 16 slice PET CT system. The pericardium showed intense uptake of FDG in the anterior, inferior and right lateral walls. The anterior wall showed FDG avid thickening measuring 10×81mms standardized uptake value (SUV) 7.74. The inferior wall of pericardium showed thickening of 107×13mms with SUV value of 12.07. Few mediastinal lymph nodes were noted as follows: subcarinal node 17×13 mms SUV 3.86, left internal mammary node 17×6 mms SUV 2.58, right internal mammary node 8 mms SUV 2.81, left paratracheal 10 mms SUV 1.80, right paratracheal 10 mms SUV 3.24. Left supraclavicular node 19 mms SUV 2.53. Right level IV neck node 16 mms SUV 2.26 , WBC 7800/ µl (6000-10000/µl); platelets 414000/ µl (150000-450000/ µl); LDH (lactate dehydrogenase) 200 U/L (100-250); Blood Widal test excluded enteric fever. Sputum for AFB (acid fast bacilli) was negative for tuberculosis. Sonography showed bilateral pleural effusions, small pericardial effusion. There was no evidence of deep vein thrombosis on color doppler scan. FDG PET CT was performed using 7.7 mCi of SUV 2.26 . BilaterSUV 2.26 . CardiacSUV 2.26 . In view of these findings a diagnosis of granulomatous disease involving the pericardium was made. Serum ACE (angiotensin converting enzyme) was recommended. The value was 72 U/L . Tuberculin test was negative. Histology  showed non- caseating Granulomas, multinucleated Langhan’s giant cells and lymphocytic infiltrates . Steroids and empirical antitubercular treatment were initiated. Myocardial biopsy was not performed as FDG PET CT of myocardium was normal. The term sarcoidosis was introduced in 1899 by Caesar Boeck to describe skin lesions caused by epithelioid cells with pale nuclei and few giant cells. Due to its resemblance to sarcoma, he called these benign sarcoid of skin . Propionibacterial and mycobacterial DNA and RNA have been identified using PCR technique. Antibodies to mycobacterium tuberculosis have been detected in serum samples of patients with sarcoidosis . Sarcoidosis may affect any organ in the body. Involvement of lungs may cause dyspnea, cough, wheezing, chest pain. Uveitis may cause blurring of vision or blindness. Eye pain, photophobia, redness may also occur secondary to eye involvement. Neuro-sarcoidosis may present as cranial nerve palsy, headache, ataxia, cognitive dysfunction and seizures. Parotid gland enlargement in sarcoidosis is also known as Mikulicz syndrome. Musculoskeletal involvement includes sarcoid arthropathy, Lofgren syndrome . Heerfordt’s syndrome . Fever, weakness, weight loss may be general symptoms. Hypercalcemia and/or hypercalciuria is found in considerable number of newly diagnosed patients of sarcoidosis . Pulmona Myocardial sarcoid may present with acute left ventricular failure, tachyarrhythmias, conduction disturbances, valvular insufficiency or sudden death . Rarely  A study showed myocardial involvement in 25% of patients with sarcoidosis in the USA, and accounted for as many as 13–25% of deaths due to sarcoidosis . InvolveLeft ventricular free wall, septum and conducting system are involved in that order of frequency. Pericardial involvement results in restrictive pericarditis. It may rarely cause constrictive pericarditis . Negative Tuberculin test aids in distinguishing sarcoidosis from tuberculosis. Allergy to tuberculin skin test was observed in 87% of cases of sarcoidosis. A positive test requires extensive searched for tubercular focus . 67Ga citrate is an age-old radioisotope that localizes to the transferrin receptors of iron and lactoferrin of macrophages (67Ga citrate has been reported to localize in viral pericarditis, post aortocoronary bypass pericardial inflammation (rophages . As sarcrophages , 17. 67Gammation . There iammation .67Ga-citrate uptake are lymphoma, amyloidosis, atrial thrombus, myocarditis, systemic sclerosis (Other conditions with clerosis . FDG PET CT is a sensitive method to detect foci of inflammation. Sarcoid granulomas express glut-1 receptors. Consequently, there is increased FDG uptake . FDG PET PET has the advantage of imaging the whole body distribution of disease, which is difficult with MRI. Plain and dynamic contrast MRI provides non-invasive assessment of the heart for evaluation of cardiac sarcoidosis. MRI gives information about scar, oedema, perfusion defects and abnormal biventricular function. Late Gadolinium enhancement along the ventricular walls is the typical finding of myocardial sarcoidosis. MRI without PET risks failure to diagnose active myocardial inflammation seen early in the disease until it results in fibrotic changes which occur late. T2-weighted MRI shows inflammation of pericardium in form of edema but is less sensitive than PET, and also inflammation in areas of fibrosis is not easily detected . MRI has the advantage of no ionizing radiation, but it is contraindicated in patients with implanted pacemakers or defibrillators unless they are MRI compatible. Also care is required in the use of gadolinium contrast in patients with impaired kidney function. Pericardial involvement is seen as irregular and patchy pericardial thickening with enhancement on contrast MRI. There is a case report in a Japanese journal of pericardial tuberculosis presenting with fever .Pericard Corticosteroids is the mainstay of therapy for patients with cardiac sarcoidosis as it prevents left ventricular remodelling when myocardium is involved and improves survival . Pericar Primary pericardial sarcoidosis is a rare entity. PET scan aids in detection of pericardial inflammation to target biopsy. It also helps to detect inflamed lymph nodes that could be alternate site for biopsy to arrive at a diagnosis. Our case of pericardial sarcoidosis presented with PUO. Despite a diligent search, we were unable to find a report on pericardial sarcoidosis presenting with PUO.


Unfortunately, programs cannot prevent trainees from engaging in cheating behaviours such as content sharing, and little is known about the impact of re-using items.To alleviate some of the burden associated with the development of novel quality questions on a regular basis, medical education programs may favour the use of item banks. This practice answers the real pragmatic need of having to create exams We conducted an exploratory descriptive study to assess the effect of repeated use of banked items within an in-house assessment context. The difficulty and discrimination coefficients for the 16-unit exams of the past 5 years  were analyzed using repeated measure ANOVAs.M = 79.8% for the first use of an item, to a mean difficulty coefficient of 85.2% for the fourth use) and discrimination coefficients decreased significantly with repeated uses .Difficulty coefficients increased significantly (The results from our study suggest that using an item three times or more within a short time span may cause a significant risk to its psychometric properties and consequently to the quality of the examination. Pooling items from different institutions or the recourse to automatic generated items could offer a greater pool of questions to administrators and faculty members while limiting the re-use of questions within a short time span. The re-use of written exam questions answers pragmatic needs in many health professions educational programs. However, this practice puts programs at risk for questions and exam content being leaked from one cohort of examinees to the next cohorts. We examine here the concrete effect on question difficulty and discrimination of re-using written exam questions over a 5-year period. Our results suggest the practice is feasible when limiting the number of times a question is re-used within that time span.The increased interest in competency-based education (CBE) and programmatic assessment has had an effect on assessment practices within medical education. One of those consequences is the increase in assessment frequency to provide feedback regularly –4. A freThe development of assessment that appropriately and purposefully assesses students’ knowledge and skills, provides feedback, and that yields reliable scores, challenges even the best educators –7. Even To alleviate some of the burden associated with the development of novel quality questions on a regular basis, medical education program administrators and faculty members may favour the use of item banks . UsuallyThe use of banked items creates, however, a risk of item disclosure. The known use of banked items in assessment often leads to strategies such as content sharing –14—betweWhile there are limited studies about the impact of item disclosure in the context of classroom-based assessment, several studies have tackled the issue of item disclosure in the context of repeated exposure to the same items for repeat examinees on certification or licensure exams , 22–26. Park and Yang  argue agThe aim of this study was to assess the effect of repeated use of banked end of unit exam questions on their psychometric properties (difficulty and discrimination indices) over a 5-year period in a Canadian undergraduate medical education (UGME) program.We conducted an exploratory and descriptive study to investigate the effect of re-using written exam questions on their psychometric properties. The study was conducted within a Canadian undergraduate medical education program that uses a problem-based learning (PBL) approach. The curriculum has been organized in sequential blocks. During the first two years, students are exposed to 16 separated system-organ blocks in addition to a block dedicated to biomedical knowledge and a block serves as introduction to their future profession as physicians. These blocks are almost exclusively taught through PBL and mobilize 25–35 tutors per block. Each unit has a formal written and oral assessment.At the time when the study was conducted, the Canadian Tri-Council policy statement on ethical conduct for research involving humans stated that consent was not required from participants for a secondary analysis of anonymized data.Written exams for the 16 system-organ PBL blocks within the UGME program are comprised of some banked  and some new, MCQ and SAQ. New questions (or significantly modified questions) are written by a faculty member and are subsequently vetted by two faculty members for appropriateness, purposefulness and difficulty . Questions are not pretested. Questions are vetted after each administration, any significant change  would make that question ‘significantly modified’ and categorized as a new question. There is no washout period for questions, meaning a question could be used two years in a row. Block exams vary in length and ratio of new/used items, as there are no definite standards of practice. More specifically, the written exams can comprise about 50 to 90 questions, with 25 to 60% of re-used questions. MCQ answers receive either 0 or 1 according whether the candidate correctly identifies the appropriate answer. SAQ are marked on a scale of 0 to 1, given a weighted mark, depending on the number of sub-questions answered. For example, if the SAQ requires a list of three diagnoses, each correct diagnosis would be given a 0.33 mark.n = 972 students)). In that period, and for all cohorts and exams combined, 1,629 were identified as re-used questions meaning that they were used more than once in that period. These questions were thus included in our analysis. In other words, we pooled data from 90 different exams, with 16 exams administered to each of our five cohorts over the 2.5 years of their pre-clinical training.We collected data for the 16 different block exams administered to five different cohorts  for the different number of times used  as a within-subject comparison (independent variables). In other words, using the number of times a question was used as a within-subject comparison (where questions are treated as the subjects of interest), we investigated the impact on two dependent variables, that is, the difficulty and the discrimination coefficients. A total of six ANOVAs were computed. Analyses were done using SPSS 21 .Questions were only categorized in one of the three categories  as illustrated in Fig. M = 0.16, and 0.13, for 2nd and 3rd uses respectively).We observed an increase in question difficulty with repeated use, and independent of the number of uses. The overall mean difficulty coefficient of 79.8% for the first use of a question, to an overall mean difficulty coefficient of 85.2% for the fourth use of a question within a 5-year period . We observed a decrease in discrimination coefficients with repeated use, from an overall mean discrimination coefficient of 0.16 on the first use to an overall mean discrimination coefficient of 0.14 for the last use (p < 0.001), three times  or four times . The decrease in question discrimination was significant for questions that were used three times  or four times .Tab. de novo at each administration. Re-using items, however, comes with potential disclosure risks which can be a real threat to the validity of assessment data interpretation. This study analyzed the psychometrics properties of 1,629 re-used questions within a 5-year span in the context of a PBL UGME program at a Canadian University.The use of in-house item banks answers a real pragmatic need of having to create exams We observed a change in question property with an increased use of the question. More specifically, items which were re-used over a 5-year period became decreasingly difficult and less discriminating over time. The increase in difficulty coefficients, or the appearance of questions getting easier, may have several underlying contributors. As suggested earlier, item repeat increases the risk of item disclosure and there is evidence in the literature –14 for sWhether the observed change in psychometric properties is pedagogically significant—and can thus impact the validity of assessment data interpretation—is debatable. To assess the pedagogical impact of these results, one should consider what an overall 6% increase in difficulty coefficient and a 0.02 drop in discrimination coefficient represents. In most Canadian universities, the grading system translates a score (number) to a letter with an increase  at each 5% increments . Thus, creating an exam with frequently re-used banked questions could potentially increase students’ scores and consequently their final grade . The effect of decrease in discrimination may not be as impactful; it seems the discrimination coefficients were only minimally influenced by question re-use.n = 83) was used four times, and no question was used more than four times. We cannot posit on what happens when a question is used more than four times. We pooled data from 90 different exams (18 blocks × 5-year span) and thus had a large sample of re-used questions. Moreover, each exam is comprised of questions written by 2 to 3 different faculty members. We could not access frequency for MCQ options, which also would have been very interesting data to study. If options become less attractive through time, we could speculate about content/questions leakage from one cohort to the next. Although the results of this study cannot generalize beyond similar contexts, the significant changes in psychometric properties should not be overlooked.This study has limitations. The study was conducted within only one program, for a limited time span (5 years), where only a small sample of questions  can impact the question’s psychometric properties. Pooling questions from different institutions could be an interesting way to increase item-bank size and thus access to questions with known psychometric properties, while limiting the re-use of a specific question within a short time span, as also suggested by Gotzman and colleagues . Gierl a