Superlubricity of graphite and graphene has aroused increasing interest in recent years. Yet how to obtain a long-lasting superlubricity between graphene layers, under high applied normal load in ambient atmosphere still remains a challenge but is highly desirable. Here, we report a direct measurement of sliding friction between graphene and graphene, and graphene and hexagonal boron nitride (h-BN) under high contact pressures by employing graphene-coated microsphere (GMS) probe prepared by metal-catalyst-free chemical vapour deposition. The exceptionally low and robust friction coefficient of 0.003 is accomplished under local asperity contact pressure up to 1 GPa, at arbitrary relative surface rotation angles, which is insensitive to relative humidity up to 51% RH. This ultralow friction is attributed to the sustainable overall incommensurability due to the multi-asperity contact covered with randomly oriented graphene nanograins. This realization of microscale superlubricity can be extended to the sliding between a variety of two-dimensional (2D) layers. Superlubricity can be unstable in graphene systems, especially under high applied loads. Here the authors use microspheres uniformly coated by graphene to measure friction between 2D materials and show that superlow friction is preserved for long periods of time under high loads and various atmospheres. And the sliding happens between the graphene nanoscrolls and randomly arranged diamond-like carbon atoms, forming an incommensurate contact2 microsphereIn this paper, we conceive a method to directly measure the friction between 2D materials under high applied normal loads by using graphene-coated microsphere (GMS). Graphene-coated commercial atomic force microscopy (AFM) sharp tip has already been developed for studies of electronic properties of materials252 microspheres by an atmospheric CVD system. Schematic illustration of the preparation process is shown in 2 microspheres were firstly dispersed in ethanol, and then transferred onto quartz plate by capillary glass tube. The quartz plate together with the SiO2 microspheres were placed at the centre of quartz tube in a CVD furnace. The CVD growth procedure involves pre-heating, substrate annealing and high-temperature growth in a gas atmosphere consisting of methane, hydrogen and argon. This method avoids the participation of metal catalyst and complicated post-growth transfer processes. The detailed growth processes are described in the Methods section. After the CVD growth process, the graphene-coated SiO2 microsphere was attached to a tipless silicon cantilever with ultraviolet (UV) light solidify glue to yield a GMS probe.The graphene film was grown directly on the 8 μm SiO2 microsphere. The microsphere surface is covered with a continuous and homogeneous graphene film. The morphology of the graphene film is relatively rough, ascribing to the original rough surface of the bare SiO2 microsphere substrate. The existence of graphene film on SiO2 microsphere is firstly verified by Raman spectroscopy, as shown in −1, respectively. The ratio of the relative intensity between 2D and G peak (IG/I2D) indicates a multilayer graphene (MLG) structure on the microsphere−1 shows structural disorder or effects of grain boundaries. An average grain size of approximately 160 nm is estimated by atomically resolved AFM measurement for graphene films grown on flat SiO2 substrate  basal plane on the microsphere is assumed to be beneficial to reduce sliding friction due to the low shear strength between graphene layers.Cross-sectional high resolution transmission electron microscope (HRTEM) was employed in order to further investigate the structure and thickness of the graphene film on the microsphere. Au and Pt films were firstly sputtered on the region of interest to prevent the graphene film from further damage during the TEM specimen preparation. The sample was then picked out from the top of the microsphere. The four different layers of the HRTEM image in 2/SiO2, SiO2/Gsub, MLG/SiO2 and MLG/Gsub, where MLG represents multilayer graphene grown directly on SiO2 microsphere, and Gsub represents the monolayer graphene firstly grown on Pt foil by CVD process and then bubbling transferred onto the flat SiO2/Si substrateThe friction measurement was mainly conducted by MFP3D AFM in lateral force mode under ambient atmosphere, as well as by NT-MDT AFM with an environmental chamber under dry nitrogen and different humidity. Friction force was measured by the half width of the lateral force loop under different applied loads, as shown in FL stands for the friction force, μ represents the slope of the friction versus normal force curve, which is defined as friction coefficient here, FN is the applied normal force and F0 is the offset friction force when the applied load is 0. We found that even for the MLG/HOPG system, the offset force is positive (around 1∼3 nN) at zero applied load (where ied load . As can μ differs from one another. The friction coefficient of the bare SiO2 microsphere probe sliding on SiO2 substrate (SiO2/SiO2) is as high as 0.6. However, with the presence of monolayer graphene on the substrate (SiO2/Gsub), the friction coefficient decreases markedly to 0.22. Similarly, with the growth of MLG on the SiO2 microsphere (MLG/SiO2), the friction coefficient decreases to 0.1. When both sliding surfaces are coated with graphene, that is, the GMS slides against graphene on substrate (MLG/Gsub), where sliding happens between the graphene layers, the friction coefficient reaches as low as 0.025. This experimental setup enables the direct measurement of interlayer sliding friction between 2D materials, which shows excellent frictional performance of the graphene/graphene tribo-pair.By comparing different tribo-pairs, we find that not only the absolute value of the friction force, but also the slope of the fitting line 2 microsphere probe, freshly cleaved HOPG  and nature graphite were also used as the mating material. The friction forces of the two different tribo-pairs: SiO2/HOPG and MLG/HOPG with a function of applied normal load, are compared in 2/HOPG, the friction coefficient extracted from the slope of the fitting line is 0.046, compared to that of 0.22 for the SiO2/graphene tribo-pair, indicating a better frictional performance on HOPG than on CVD graphene. Two possible reasons may explain the phenomenon: Firstly, the freshly cleaved HOPG sample is usually defect-free in a relatively large single crystalline domain, as compared to the possible defects generated during the growth and transfer processes of the CVD graphene. Secondly, the bulk HOPG is atomically smooth and free from roughness and puckering effects which reportedly cause a friction rise in single- and few-layer graphenesub substrates are further discussed by using mechanical exfoliated monolayer graphene on SiO2 substrate as a reference in To further investigate the friction properties of the graphene-coated SiO2/HOPG , which is verified by MD simulation in Furthermore, we have also obtained superlow friction coefficient between GMS and graphite under different experimental conditions. As shown in 2/SiO2 tribo-pair exhibits the highest friction coefficient owing to the strong covalent bond interactions between the SiO2 surfaces. The presence of graphene on the substrate can significantly reduce surface dangling bonds at the SiO2/SiO2 interface, thus leading to a relatively lower friction. It is worth noticing that while the sliding happens between graphene and SiO2 for both MLG/SiO2 and SiO2/Gsub tribo-pairs, the friction coefficient of MLG/SiO2 is less than half of the SiO2/Gsub. This is at the first glance out of expectation, as the MLG grown on microsphere is rough, polycrystalline as indicated by the SEM, Raman spectra results, while the transferred CVD graphene on the flat substrate is monolayer with large grain size. To figure out this counterintuitive result, Raman spectroscopy measurement of the scanned area on the SiO2 substrate after sliding with GMS was conducted  on the substrate. Afterwards, the shear plane shifts from graphene/SiO2 to between the graphene layers, which accounts for the lower friction. Nevertheless, for SiO2 microsphere sliding against monolayer graphene, it is difficult for the monolayer graphene on the substrate to transfer onto the SiO2 microsphere unless the graphene is worn out. The comparison of SiO2/Gsub and MLG/SiO2 tribo-pairs proves a relatively smaller friction between the graphene layers, thus the coating of MLG on the SiO2 microsphere can offer a better tribological performance. For the MLG/Gsub tribo-pair, the graphene coatings on both sides provide a shear plane between graphene layers with weak shear strength, giving rise to the lowest friction of the four tribo-pairs.The comparison of friction coefficient for different tribo-pairs shows the excellent friction reduction performance of graphene. SiOcted see . The sca2 microsphere can significantly eliminate the dangling bonds, leaving predominately weak van der Waals forces at the sliding interface, which has been assumed to be an important reason for the lubricity of 2D materials.The most impressive result of GMS probe is the achievement of superlubricity when sliding against HOPG or nature graphite. This superlow friction can be ascribed to the following reasons: the freshly cleaved HOPG surface is single-crystalline with a grain size from several tens micrometers to several millimeters, and the coating of graphene films on the SiOθ1=6.03°, θ2=13.09°, θ3=21.04°, θ4=26.93° as denoted in θ1=θ2=θ3=θ4=0°) and strict lattice match with the substrate, all the other contact geometries were kept the same for the curved graphene flakes in order to preclude other affecting factors, such as contact area and pressure. As shown in Furthermore, besides interlayer van der Waals interaction, the interfacial atomic registry also plays an important role in superlubricity. Based on the measured mechanical properties and the surface morphology of the microsphere, numerical simulation is conducted to calculate the contact properties between GMS microsphere and HOPG  Ar was flushed into the quartz tube to clear air trapped in the system. The furnace was then heated up to the targeted temperature 1,130 °C under a constant pure hydrogen gas flow of 200 sccm. When the furnace was heated to the targeted temperature, the H2 gas flow and Ar gas flow were tuned to 50 sccm simultaneously, and a CH4 gas flow of 6.3 sccm was introduced into the system for 2 h to initiate graphene growth. After finishing the CVD growth process, the furnace was opened to allow the system cooling rapidly to room temperature.SiO2 microsphere probe was carried out under MFP3D SA , with a camera lens and a light path underneath the transparent quartz plate, which is convenient for precisely locating the cantilever and the SiO2 microsphere. Graphene-coated microsphere was attached to AFM cantilever (Nanosensor TL-FM) with UV light solidify glue, and then exposed to UV light for 20 min.The preparation of SiOThe TEM cross-sectional sample was prepared by focused ion beam (FIB), picked out from the top of the microsphere. Au film was firstly sputtered on the graphene sample and Pt film was deposited subsequently as protection layers. The deposition of Au and Pt films, aiming to protect the interested region during FIB milling, was under low energy to prevent damage to the lamellar structure of MLG.The Raman spectra of GMS was collected on Horiba HR800 with a laser wavelength of 514 nm and × 50 objective lens under ambient conditions, and the laser spot size was 1 μm. The morphology of GMS was observed by SEM (FEI Quanta 200 FEG). TEM (JEOL 2011) experiment was conducted to observe the clear lamellar structure of graphene.The friction tests were conducted by MFP3D SA (Asylum Research) AFM in lateral force mode under ambient atmosphere, at a temperature of 26±2 °C and with a relative humidity of 18±3%. The friction tests under different atmospheres were conducted by NT-MDT AFM with a chamber to control the atmosphere up to 51% relative humidity.−1. Friction force was measured by the half width of the lateral force loop evaluated for 16 scan lines under each load, and the normal spring constant of the cantilever was calibrated using the thermal noise method. All the experiments presented in −1. Bulk h-BN (from XFNANO) was used to evaluate friction between heterostructure of graphene and h-BN.The normal spring constant of the microsphere probe prepared by the tipless cantilever (Nanosensor TL-FM) is 2.5±0.5 N m−1 for graphene-coated SiO2 microsphere probe and 79.09 μN V−1 for bare SiO2 microsphere probe.In order to derive the lateral force from the voltage signal, the lateral spring constant of microsphere probe was calibrated using a diamagnetic lateral force calibrator−1 along x direction through a spring with a stiffness of k=10 N m−1, which was used to mimic the compliance of an AFM systemThe atomistic configuration consisted of one graphite substrate with four graphene layers and altogether 67,488 carbon atoms where the atoms in the bottom layer were kept fixed during the simulation, four graphene wrapped diamond bumps (cutting from diamond nanospheres) to simulate the nanoscale asperities with a diameter of 10 nm and altogether 16,780 carbon atoms, and also one upper base to simulate the bulk microsphere with 10,588 carbon atoms. The size of simulation box is 243 Å × 183 Å × 36.5 Å. The graphene flakes were wrapped on the asperities through Leonard-Jones (LJ) potential to avoid possible wrinkling and excessive strain. The AIREBO potential was used to describe the interatomic interaction within and between the graphene flakes and graphite substrateThe data that support the findings of this study are available from the corresponding authors on request.How to cite this article: Liu, S.-W. et al. Robust microscale superlubricity under high contact pressure enabled by graphene-coated microsphere. Nat. Commun.8, 14029 doi: 10.1038/ncomms14029 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Notes, Supplementary Methods and Supplementary References.Supplementary video 1 is the side view of the simulation system showing that a water droplet is repelled away from the contact region when a graphene flake covered asperity (not shown) is loaded on a graphite surface.Supplementary video 2 is the top view of the simulation system showing that a water droplet is repelled away from the contact region when a graphene flake covered asperity (not shown) is loaded on a graphite surface.Supplementary video 3 is the side view of the simulation system describing that a water droplet is split into several smaller droplets and squeezed out of the contact area when a graphene flake covered asperity (not shown) is loaded and sliding on a graphite surface.Supplementary video 4 is the top view of the simulation system describing that a water droplet is split into several smaller droplets and squeezed out of the contact area when a graphene flake covered asperity (not shown) is loaded and sliding on a graphite surface.Supplementary video 5 is the side view of the simulation system showing the sliding process of the asperity surrounded by small water droplet and then encountering a large water droplet.Supplementary video 6 is the top view of the simulation system showing the sliding process of the asperity surrounded by small water droplet and then encountering a large water droplet.


The aim of our study was to assess current practice patterns and attitudes towards diagnosis and management of idiopathic pulmonary fibrosis (IPF) patients in Latin America.A Cross-sectional survey was developed and up to 455 physicians were enrolled. We used a rigorous method of validation using the translated version of the AIR Survey.Mean age was 47.5 years (SD 12.6) with 20.4 years (SD 12.3) of practice. In around 30% of physicians were reported access to radiologist, pathologist and multidisciplinary team. Despite almost all physicians reported that (ATS/ERS/JRS/ALAT) guidelines are useful, half of them prescribed corticoids for treatment of disease. Most respondents (69.9%) reported cough as the presenting symptom. Around 80% considered IPF to be an important clinical disorder, and felt that identifying patients at risk for IPF was important or extremely important. However, only 59.7% felt confident in managing patients with IPF, and similar numbers (60.8%) felt confident about their knowledge. Pulmonologist have more confidence and management of IPF that no pulmonologist.The results of this survey of Latin American physicians could help to fill gaps regarding awareness, management and treatment of IPF and improve earlier diagnosis of IPF.The online version of this article (10.1186/s12890-017-0569-1) contains supplementary material, which is available to authorized users. Idiopathic Pulmonary Fibrosis (IPF) is characterised by chronic, progressive and irreversible decline in pulmonary function. Clinical symptoms and natural history are unpredictable and vary in severity . BecauseUsing narrow case definitions, the annual incidence of IPF in the US is estimated to be 6.8–8.8 per 100,000. IPF incidence increases with age and is higher among males. In recent years the incidence appears to be increasing .A retrospective longitudinal study suggested that median survival of IPF patients is 3–5 years following diagnosis [Confirmation of the diagnosis requires exclusion of other causes of interstitial lung disease (ILD), along with the presence of the characteristic usual interstitial pneumonia (UIP) indicated by honeycombing on high resolution computed tomography (HRCT). In those without definitive radiological UIP, a surgical lung biopsy revealing characteristic findings of UIP is necessary for conclusive diagnosis , 7..The 2011 American Thoracic Society/ Japanese Respiratory Society/ Latin American Thoracic Society (ATS/ERS/JRS/ALAT) guidelines call for a multidisciplinary team (MDT) approach to IPF, in which information from several sources is combined and evaluated . In pracA European survey of IPF management suggested that physicians may benefit from periodic education and update regarding IPF management . In someIn Latin America, where information is scarce, there is a clear need to standardise the approach to diagnosis and treatment of IPF patients.The aim of our study was to assess current practice patterns and attitudes towards diagnosis and management of IPF patients in Latin America, to compare specifically pulmonologists and non-pulmonologists related to IPF management.We conducted an anonymous cross-sectional survey, in which physicians evaluated themselves regarding idiopathic pulmonary fibrosis. The original questionnaire  consisted of 28 questions relating to the clinical management of patients with IPF [Two of the investigators translated the AIR survey to Spanish. After that, the Spanish-language version was translated to English by a third investigator who did not know the original version of the AIR Survey. The back-translated English-language version of the new Spanish-language questionnaire was compared with the original English-language version. Each item on the back-translated English-language version was ranked by 30 individuals who were bilingual and independent of the study team for comparability and similarity of interpretability with the same item on the original English-language version. Any translated item with a mean score > 3 (seven was the worst agreement and one was the best agreement) was formally reviewed and corrected. The revised item was then translated back to English and compared again with the original English-language version of that item. This process continued until the mean scores for each item indicated a valid version  .The target population included physicians from Ecuador and elsewhere in Latin America who attended the following meetings between November 2015 and October 2016: the VII-VIIIth International Meeting of Allergy and Respiratory Medicine, Guayaquil, Ecuador; and the XIIth Latin-American Expert Forum Santiago, Chile. The inclusion criterion was having received a diploma as a medical doctor. Potential candidates were asked if they often managed patients with IPF. If they answered in the affirmative, they were eligible for the study, and a self-administered survey was delivered. This study was performed in accordance with the Declaration of Helsinki. This human study was approved by Comité de Ética en Investigación en Seres Humanos - Clínica Kennedy. All adult participants provided verbal consent to participate in this study and this was approved by our IRB and was because survey was anonymous and no interventions were conducted. The verbal consent process was recorded/documented by the moment in which physician completed the questionnaire.The first 28 questions of the AIR survey assessed the current diagnostic and management of IPF, including pharmacological therapy by physicians experienced in IPF and management of comorbidities. Furthermore, we included two “attitude questions” regarding the importance of IPF as a clinical disorder and IPF diagnosis, and three attitude questions regarding the physician’s confidence in diagnosing, managing and knowledge about IPF. These “attitudes and confidence questions” were scored on a five-point Likert scale.p-value <0.05 was significant.We used mean and standard deviation (SD) for age, years of practice, and for number of patients with IPF attended according to their age and forced vital capacity (FVC). Percentages (%) were calculated for gender, nationality, location of medical’s office, pulmonologist, access to pathologist, radiologist, multidisciplinary team, perception of IPF guidelines, risk factors and onset of disease, treatment, monitoring progression of IPF and managing comorbidities, and attitudes towards disease. Attitudes towards disease were summarized as very important (very important and extremely important) and very confident (agree and strongly agree). Mann-Whitney U tests and chi square were used for comparisons among pulmonologists and non-pulmonologists and number of patients with IPF attended according to their age and FVC. Crude logistic analysis were performed among pulmonologists (non-pulmonologist as reference category) and access to pathologist, radiologist, multidisciplinary team, perception of IPF guidelines, risk factors and onset of disease, treatment, monitoring progression of IPF and managing comorbidities, and attitudes towards disease. Statistical tests were performed using SPSS version 13 . A Up to 455 physicians were enrolled. The majority were male (55.4%). In total, 23.7% reported being a pulmonologist. Mean age was 47.5 years (SD 12.6) with 20.4 years (SD 12.3) of practice.n = 340) reported having cared for an IPF patient in previous year. Among these physicians, 38.6% were female, attending in private settings (48.4%), and one third (29.7%) were pulmonologists. Most of participants were from Ecuador (67.9%), followed by Chile (9.7%) and Peru (8.8%) .On average, 13.8 (SD 21.4) IPF patients were cared for in the previous year. Physicians reported the highest average number of IPF patients in the age group 66–80 years old , with a forced vital capacity (FVC) ranging from 50%–70%  (Table Approximately one third of survey respondents reported access to pathologists (27.8%), radiologists (39.4%), and multidisciplinary teams (26.9%). Pulmonologists reported greater access to radiologists  and pathologists . Access to multidisciplinary teams was more common in pulmonologists (41.1% vs 20.8%) compared with non-pulmonologists (p < 0.001).p < 0.05). Up to 49.5% were aware that average time between symptom onset and diagnosis was ≤6 months, with no differences among specialties.ATS/ERS 2011 guidelines were considered useful by almost all respondents (93.7%). No differences were found among among physician specialties . A total of 18.9% of respondents reported that 0–10% of their IPF patients smoke. Pulmonologists reported higher rates of attending patients who smoking  of respondents reported that their patients had visited one or two physicians prior to diagnosis, with no differences among specialties.Most respondents (69.9%) reported cough as the presenting symptom. This result held with pulmonologists and non-pulmonologists , and was similar among countries. In addition, dyspnoea was frequently reported (54.6%), more often by pulmonologists ). “Velcro” breath sounds were considered to be the first sign of IPF in 27.9% of respondents, more frequently in pulmonologists .p < 0.001).Up to 92.8% of respondents reported obtaining family history. In total, 97.9% inquired about occupation, while 18.4% pursued genetic causes. Up to 22.3% of respondents reported managing mild and moderate IPF patients themselves, without collaboration. This finding was more frequent among pulmonologists (35.0% vs 17.2%)  to make and confirm the diagnosis, to manage comorbidities, to develop a monitoring plan, and to monitor efficacy and tolerability of medication. Differences were not found among physician specialties.Supplemental oxygen was prescribed most often 71.2%), followed by corticosteroids alone (48.0%), N-acetylcysteine (NAC) alone (29.1%), and pirfenidone (17.0%). Higher chances of supplemental oxygen prescription  and pirfenidone  were observed in pulmonologists  (Table The most common metrics for assessing progression of disease were dyspnoea 63.7%), followed by FVC (62.1%). Diffusing capacity (DLCO) was used less commonly (24.8%). Similarly, only 28.4% of respondents reported using the 6-min-walk test (6MWT). Pulmonologists reported higher chances of employing for most of these tests . GERD was considered very important to manage by 48.5% of respondents, but was treated systematically by only 32.3% (37.8% by pulmonologists vs 29.8% by non-pulmonologists). Non-pulmonologists considered managing GERD as very important (55.6%) more frequently than pulmonologists (45.3%).In total, 64.1% of respondents reported always investigating for gastroesophageal reflux disease (GERD) , followed by proton pump inhibitors (PPI) (69.3%) for treatment of GERD. PPI was more commonly prescribed by pulmonologists ). H2 blockers were prescribed by one quarter of respondents (23.9%). This non-recommended treatment was recommended more commonly by non-pulmonologists ).p < 0.05).The most frequent assessed comorbidity was cardiovascular disease (9.1%), followed by GERD (8.6%), and lung cancer (7.4%). Lung cancer was assessed more frequently among pulmonologists  was treated in 55.0% of patients. Sildenafil was used by half of respondents (52.5%), more frequently by pulmonologists ).p < 0.001)  felt confident about their knowledge (regarding diagnosis and management) of IPF. Pulmonologists reported highest rates of importance and confidence (1) Table .Table 4CIn our study, approximately one quarter of Latin American respondents reported access to a pathologist or a multidisciplinary team in their professional network, as compared with European participants . Meanwhile, European physicians reported greater access to a radiologist 85%), in our continent this access is only available in one third of physicians [5%, in ouEarlier diagnosis of IPF has become more relevant since controlled clinical trials have demonstrated a reduction in the rate of decline of FVC , 15. AlmOur respondents reported that the most common presenting symptom was cough (69.9%). This result was similar across specialties. This result differs from European reports , becauseOur respondents reported that 22% of their patients smoked, a substantially smaller percentage than reported in the European survey (60%) . This fiBecause IPF is a disease with a variable clinical course, it is important to identify factors that may help predict prognosis. Published guidelines recommended such tests as FVC, DLCO and 6MWT , 17. TheOnly 10% reported assessing GERD in IPF patients. Half of respondents treated PH patients with sildenafil, in contrast to the AIR survey, in which only a small proportion of respondents (4%) would regularly treat PH. .We predicted that IPF recommendations would be followed by our respondent group because almost half of participants reported they felt IPF guidelines were useful. This was not necessarily the case. First, corticosteroids were frequently prescribed, in combination with immunosuppressive therapy or NAC, contrary to guidelines. A similar discrepancy was found in an Australasian study . HoweverApproximately one third of respondents prescribed no specific treatment for IPF, similar to the result found in European surveys , 9. PirfThere are some disagreements between IPF guideline recommendations and confidence in managing patients with IPF. These discrepancies may reflect a degree of self-protection among respondents.Our results are comparable to the AIR study because respondents manage IPF patients with similar levels of severity . EstabliWe suggest that optimal physician management of IPF helps to identify factors that substantially affect patient outcomes. Updated IPF guidelines in local languages could lead to improved collaboration among primary care physicians, pulmonologists, specialised centres, pharmacists and other stakeholders involved in IPF management.Our study has several limitations. First, this was a cross-sectional survey of physicians attending conferences. Therefore, we cannot infer causation from any of the associations we observed. We cannot generalise our results to all Spanish-speaking physicians because medical education regarding IPF might differ in other Spanish-speaking countries in important ways. Furthermore we have few physician from other Latin-American countries and we cannot generalize our result from all Latinamerica. Physicians who travel to medical meetings are likely to attend educational programs routinely, and therefore these physicians might have access to more updated medical knowledge than the larger group of general practice physicians practicing in the community who do not attend continuing medical education meetings. Thus, management of IPF in the larger population of general practice physicians and pulmonologists is likely to be less optimal than was reported in our survey. Furthermore, survey was self-administered with subjective answers, that could reflect some protection bias. Finally, our study didn’t include radiologists and pathologist, who are members of MDT in diagnosis of IPF.The results of this survey of Latin American physicians could help to fill gaps regarding awareness, management and treatment of IPF especially in GP. Future studies are needed to validate this survey in Latin America and other Spanish-speaking physician populations. Also, IPF guidelines in clinical practice in Latin America could be implemented according to clinical scenario in our region. Incorporating IPF-focused educational interventions during medical school and residency training programs could help to improve management of the disease. Latin American pulmonology societies could offer more IPF sessions at continuing medical education conferences. They could also create IPF education programs for general physicians such as those that currently exist for asthma and chronic obstructive pulmonary disease.Despite high rates of awareness of international IPF guidelines, most IPF recommendations are not followed. To achieve better outcomes for our patients, we recommend improving IPF education in Latin America. Better knowledge of the disease and improved skills in generating a differential diagnosis may contribute to earlier diagnosis and treatment of IPF.


We study the nonlinear dispersion and coupling properties of the graphene-bounded dielectric slab waveguide at near-THz/THz frequency range, and then reveal the mechanism of symmetry breaking in nonlinear graphene waveguide. We analyze the influence of field intensity and chemical potential on dispersion relation, and find that the nonlinearity of graphene affects strongly the dispersion relation. As the chemical potential decreases, the dispersion properties change significantly. Antisymmetric and asymmetric branches disappear and only symmetric one remains. A nonlinear coupled mode theory is established to describe the dispersion relations and its variation, which agrees with the numerical results well. Using the nonlinear couple model we reveal the reason of occurrence of asymmetric mode in the nonlinear waveguide. At THz and far-infrared frequency range, the electrons transition of intraband dominates primarily and the metallic conductivity of Drude type makes the graphene surface plasmon be supported. Based on its unique electric and optical properties234et al.Considerable effort has been devoted to investigating the mode propagation6789111314151617181920et al.19The coupling effects of graphene plasmon have attracted wide interest. The demonstration of surface plasmon excitation in graphene based on the near-field scattering of infrared light has been reported13As was shown, graphene is a strongly nonlinear material222425262726ε2 is bounded by the graphene layers at x = ±d/2 with the surrounding dielectric (ε1 = ε3). According to the Kubo formulaσL contains the interband and intraband transition contributions. In the THz and far-infrared frequency range, the intraband transition dominates the linear conductivity of graphene which can be reduced to the Drude form29The nonlinear graphene plasmonic waveguide is illustrated in e is the electron charge, μc is the chemical potential of graphene, ω is the frequency, and τ is the momentum relaxation time. This model is applicable in low temperature limit (kBT ≪ μc) at low frequency (ħω ≤ μc).where For the strong field condition, the nonlinear part of the conductivity must be considered and the total conductivity of graphene reads27Eτ is the tangential component of the electric field and σNL denotes nonlinear conductivitywhere νF = 0.95 × 108cm/s is the Fermi velocity.where z direction with a propagation constant β, the magnetic and electric field should be in the form of H = Hy±, exp (iβz ± Kxx)k0 = ω/c. According to the boundary condition, the tangential component of electric field must be continuous while that one of the magnetic field has a discontinuity of σgEz1,+,, i.e.,Considering the transverse-magnetic (TM) surface plasmon polaritons mode that propagates along x. Similar boundary condition was also established at lower boundary. The Maxwell equation gives the relation‘±’ in the subscript represents the field decrease and increase along upward direction of β,ω,Hz1,−  ≡ H0).Applying H0 on the propagation constant β at wavelength λ = 10 μm is shown in d = 100 nm, ε1 = 1, ε2 = 2.25, μc = 0.27 eV and τ = 1.5 ps. The propagation constant β is normalized by Fermi vector kF = (πn)1/2 n = 6 × 1012 cm−2. There are three modes in the nonlinear plasmonic waveguide, which are symmetric mode, antisymmetric mode and asymmetric modeDependence of the magnetic field Ez is a symmetric. Therefore, the branch I represents the symmetric mode. For branch II distribution of electric field Ez shown in For branch I the fields are plotted in σNL = 0) and by the solid curves in nonlinear case. For the linear case only symmetric and antisymmetric modes exist. The black dotted curve and the red dotted curve represent the symmetric and antisymmetric modes, respectively. In H0 = 1000 A/m) and different chemical potentials μc is given. As is shown in μc = 0.19 eV, only symmetric mode represented by the solid curve is found. It is seen from μc = 0.27 eV) leads to the intersection of antisymmetric and asymmetric modes, which is seen in μc = 0.27 eV, and to decreased initial magnetic field H0. Decrease of H0 leads to consequent reduction of nonlinearity of graphene. In this case the fold-back point of the dispersion relations moves down. In addition, as is shown in ω/μc = 0.45). The lower branch of mode I is not plotted in Next, we turn our attention to discuss the influence of nonlinearity of graphene on dispersion relation. In a1 and a2 satisfy the matrix equationIn the case of weak field without nonlinearity, the coupled graphene plasmonic waveguide shown in β1 and β2 are the propagation constants of the single layer graphene waveguide without coupling and κ is the coupling coefficient. The weak field condition of symmetric structure without nonlinearity corresponds to β1 = β2 = β0. The propagation constants of the coupled mode are defined as the eigenvalues of the matrixwhere κ = 4.3 × 10−3kF at wavelength λ = 10 μm.They could also be obtained from the mode analysis method. When the graphene’s nonlinearity is considered, the propagation constant of each single graphene waveguide becomes a function of tangential component of electric field. The dispersion of the single graphene waveguide is32β1 and β2 in β1,2(|Eτ|2) and treating a1,2 as the tangential component of electrical field in amplitude, we obtain the coupled mode theory in nonlinear casewhere a1 + γa2|2 and |a2 + γa1|2 are the total field intensity with the similar meaning to the |Eτ,+1,|2 and |Eτ,−3,|2, respectively, and γ is an empiric factor related to β and d, which is fitted from the numerical data shown in z = iβ), a1 and a2 must satisfy where |a20 = ±a10, and the third one can be only obtained numerically shown in a20 = ±a10 and a20 = f(a10) represent three branches obtained from γ ~ −0.07is shown in H0|~|a1 + γa2|ωε0ε1/Kx,1 can be used. It is found that the theoretical result consistent with the numerical one shown in The first two solutions are a20 = ±a10 leads to the symmetric increase of β1 and β2, hence, equality β1 = β2 is always established. Corresponding branches are presented by black and red curves in a10 > a20 > 0 (or a20 > a10 > 0), the former term in a20 = f(a10), we have the blue branch as shown in β1(|a10 + γa20|2)) increase caused by the nonlinearity and compensation (κa20/a10) due to the coupling.The symmetric condition of μc = 0.27 eV, 0.22 eV), the dispersion relations shows three branches, and there is a fold-back point in each branch. Continuing to increase the nonlinearity of grapheme (decreasing μc to 0.19 eV), the fold-back point disappears and there is only one branch corresponding to the symmetric mode. By introducing the nonlinear coupled mode theory, the features of the nonlinear plasmonic waveguide could be understood well. The reason for emergence of asymmetric mode is revealed. It is originated from the equilibrium of the propagation constant increase caused by the nonlinearity and the compensation due to the coupling.In summary, the coupled and dispersion properties of the graphene-dielectric-graphene structure are studied. The propagation constant is found to increase with the field intensity for both the symmetric and antisymmetric mode, whereas the antisymmetric mode splits off an asymmetric mode. When the nonlinearity of graphene is small .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Plasmonically induced transparency (PIT) in a multicavity-coupled graphene-based waveguide system is investigated theoretically and numerically. By using the finite element method (FEM), the multiple mode effect can be achieved, and blue shift is exhibited by tunable altering the chemical potential of the monolayer graphene. We find that the increasing number of the graphene rectangle cavity (GRC) achieves the multiple PIT peaks. In addition, we find that the PIT peaks reduce to just one when the distance between the third cavity and the second one is 100 nm. Easily to be experimentally fabricated, this graphene-based waveguide system has many potential applications for the advancement of 3D ultra-compact, high-performance, and dynamical modulation plasmonic devices. Electromagnetically induced transparency (EIT) refers to a quantum destructive interference phenomenon between two excitation pathways with a sharp transparency window –3. BasedGraphene, a single-layer carbon atom arranged in a honeycomb lattice, has been vigorously investigated as a promising platform for plasmonic result from its remarkable characteristics, such as strong mode confinement, low loss, and active tunability . In partIn this paper, we investigate a novel 3D graphene-based plasmonic waveguide system. This system is composed of one guided graphene waveguide (GGW) coupled to one to three graphene rectangle cavity (GRC). The multimode PIT phenomenon has been observed in our proposed system. Two transmission dips exist with one GRC while two PIT peaks appear in the dip-position of the transmission spectra with two GRCs. The increasing number of the GRC placed above the GGW introduces the multi-PIT peaks. In addition, the multimode PIT effects can be dynamically controlled by varying the chemical potential and the coupling distance. Moreover, the modulation of PIT peaks with three cavities is investigated in detail. For the advancement in 3D ultra-compact high-performance PIT devices, our work may have potential applications in the PIT-peaks modulation, slow light and optical switching.d and L, respectively. The width of the waveguide is D. Here, D and d are fixed to be 10 nm, L is 100 nm. In addition, H and h represent the waveguide-cavity coupling distance and the cavity-cavity coupling distance, respectively. Chemical potential μc is applied to the graphene waveguide and cavities. We carry out the three-dimensional numerical simulations by using the FEM [σg including the intraband and interband transition contributions [A schematic of the structure of the designed tunable peak modulation devices based on three GRCs waveguide system is shown in Fig.  the FEM . The Kubibutions :Fig. 1aμc, photon frequency ω, momentum relaxation time τ, and temperature T. Here, e represents the electron charge. In our numerical simulations, the incident wave is at mid-infrared where the influence of interband transition in graphene can be negligible. In this case, the optical conductivity can be simply given by:It depends on chemical potential k0 = 2π/λ and the intrinsic impedance of air η0 ≈ 377 Ω. The monolayer graphene support TM-polarized SPPs is only in consideration for our analysis. The dispersion relation of this TM GSPP surface wave follows the equation:βGSPP is the propagation constant of GSPPs. Defined as neff = βGSPP/k0, the effective index of GSPPs features the capacity for confine SPPs on graphene. Obviously, from the equations above, the Re(neff) increases for a fixed wavelength as the chemical potential μc decreases, which intends the GSPPs are better confined at lower chemical potential. Importantly, with a slight change in chemical potential, the Re(neff) varies greatly, which leads to the design of dynamically tunable peak modulation devices. The transmission of GSSPs with one graphene cavity satisfies the resonant condition:The following equation shows the equivalent permittivity of graphene :4\documeφ1 is the additional phase [m standing for the order of resonant mode (RM), is an integer. L is length of the cavities. Moreover, it should be noted that we assume the substrate to be air in our 3D simulations for simplification. The reason is given in the following part.Here, al phase . m standns = 1 and ns = 1.23 represent the substrate to be air and SiO2, respectively. From Fig. H is shown in Fig. As shown in Fig. λ1 = 7 μm at μc = 0.3 eV) and dip2 (λ2 = 5.65 μm at μc = 0.3 eV) is observed. Subsequently, Fig. The effective refractive index of the waveguide mode in relation to the incident wavelength is depicted in Fig. λ1 = 7 μm and λ2 = 5.65 μm can be generated in the transmission spectra in the condition that the one GRC is in resonance with the waveguide. There are, respectively, two obvious transmission PIT peaks at the two resonant wavelengths due to the near-field coupling between the three cavities, and the waveguide as depicted in Fig. Hz| with one cavity in the two resonant wavelengths are shown in Fig. λ1 = 7 μm, the third order RM exists in the GRC, while λ2 = 5.65 μm satisfies the fourth order resonant condition. At the same time, most parts of the GSPPs cannot confine to the GRC and thus propagate to the right port of the GGW. The PIT effect can be shown by the transmission spectrum with multi-cavities under the chemical potential μc = 0.3 eV in Fig. λ1 = 6.95 μm and λ2 = 5.6 μm, respectively. It indicates that two GRCs generate one PIT peak. Subsequently, we investigate the transmission properties of the waveguide system with three and even four graphene cavities. As shown in Fig. Based on the transmission characteristics of the graphene waveguide with one cavity, we investigate the properties of the system with multi-cavities. The black line in Fig. λ ∝ (2π2ℏcD/α0μc)1/2 ∝ (1/μc)1/2 [As the surface conductivity of graphene depends on the chemical potential can determine the propagation of the GSPPs, we can achieve the dynamic tunability of the PIT peak by changing the chemical potential of cavities and waveguide. The transmission spectrum with different chemical potential is shown in Fig. /μc)1/2 , 36. As C is used to describe the distance between the center of the second cavity and the center of the third one. It is apparent that the cavity coupling between the second and the third cavity is gradually weakened as C increases. Therefore, the number of PIT peaks in the two PIT windows decrease to one at C = 100 nm as depicted in Fig. z direction with different coupling distances C in Fig. λ = 5.55 μm. As shown in Fig. C is too far to confine. Thus, the peak number reduces to 1 in good consistent with the transmission spectrum seen in Fig. To further explore the influence of the rectangle cavities to the PIT peaks, we change the position of the third cavity with other parameters remain the same. Here, C can lead to the subdued cavity-cavity resonance and eventually achieve the peak modulation. We believe the proposed structures, easily to be operated, may promise the applications for tunable multiple mode filters and slow light devices.In conclusion, our work particularly has focused on the peak modulation of the multicavity-coupled graphene-based waveguide system by using FEM. One graphene cavity leads to a transmission dip while two cavities generate a PIT window in the transmission spectra. The number of the PIT peaks is always one less than the number of the cavities. Moreover, not only a blue shift can be achieved in transmission spectra with the increasing of chemical potential, but also a good linear relationship between peak wavelength and chemical potential is obtained. The increasing coupling distance


Differences in textural features or biomarkers between tumors with and without PD-L1 expression were tested using a Mann–Whitney U test. The predicted values for PD-L1 expression were examined using logistic regression analysis. The mean percentages of tumor PD-L1 expression were 6.2 ± 13.5. Eighteen tumors had PD-L1 expression ≥5%, whereas 30 tumors ≥1%. Using a 5% cutoff, the p16 staining percentage and the textural index of correlation were two factors associated with PD-L1 expression. The odds ratios (ORs) were 17.00 (p = 0.028) and 0.009 (p = 0.015), respectively. When dichotomizing PD-L1 at 1%, the p16 and Ki-67 staining percentages were two predictors for PD-L1 expression with ORs of 11.41 (p = 0.035) and 757.77 (p = 0.045). p16 and Ki-67 staining percentages and several PET/CT-derived textural features can provide supplemental information to determine tumor PD-L1 expression in HNCs.To know tumor PD-L1 expression through IHC or the FDG-PET related radiomics, we investigated the association between programmed cell death protein 1 ligand (PD-L1) expression and immunohistochemical (IHC) biomarkers or textural features of 18F-fluoro-2-deoxdeoxyglucose positron emission tomography ( Advances in the understanding of the immune system’s role in tumor development have led to malignant cells being recognized as having the ability to elude immune control through the deregulation of inhibitory signals or other specific immune checkpoints. A randomized trial investigating the inhibitors of programmed cell death protein 1 (PD-1) in recurrent HNC squamous cell carcinoma showed promising results3. Tumor PD-1 ligand (PD-L1) expression reflects an immune-active microenvironment; although it is associated with other immunosuppressive molecules including PD-1 and PD-L2, PD-L1 expression is the single factor most closely correlated with response to anti-PD-1 blockade4. Although no standardized method for PD-L1 detection or cutoff has been defined, significant associations were found between tumor PD-L1 expression, the presence of intratumoral immune cell infiltrates, and the expression of PD-1 receptor in tumor-infiltrating lymphocytes (TILs). This suggested that PD-L1 reflects an immune-reactive milieu4. In addition, recent data suggested that patients with tumors that overexpress PD-L1 have an increased chance of superior clinical outcomes after anti-PD-1–directed therapy5. To date, other than quantifying PD-L1 through immunohistochemistry (IHC), no predictors are available to identify patients with tumors that have a higher PD-L1 expression and may benefit from checkpoint inhibitors.Head and neck cancer (HNC) is the fifth most common malignancy worldwide18F-FDG PET) remains one of the most extensively used methods in the diagnostic workup of patients with various cancers. Recently, a pilot study for nonsmall cell lung cancer showed a direct association between metabolic parameters on FDG-PET and the expression of tumor-related immunity markers, suggesting a potential role for FDG-PET in characterizing the tumor microenvironment and selecting suitable candidates as checkpoint inhibitors6. However, no studies have reported the correlation between metabolic information on FDG-PET and the tissue expression of PD-L1 across various cancers. Because of the intrinsically dynamic nature of immune responses, and given the hurdles of performing new tumor biopsies aimed at investigating potential molecular predictors of checkpoint inhibitor activity in pretreated HNCs, noninvasive or minimally invasive approaches to aid patient selection are warranted. Moreover, the microenvironments of cancer tissues often present with biological heterogeneity corresponding to hypoxia, angiogenesis, or immunogenicity7. Such biological characteristics are of interest because they are associated with radiomics8. To gain greater knowledge regarding the effect of a tumor microenvironment, detected through IHC or the FDG-PET related radiomics, on tumor PD-L1 expression, we conducted this study to investigate the associations between tumor PD-L1 expression, several protein biomarkers involving the pathogenesis of hypoxia, angiogenesis, and proliferation, and 18F-FDG PET-based textural features in patients with HNCs.Among image-based modalities for noninvasive tumor assessment, positron emission tomography with 18-fluorodeoxyglucose (18F-FDG PET-computed tomography (PET/CT) for staging. All patients had normal serum glucose levels before undergoing PET/CT. This study was approved by a local institutional review board (CMUH103-REC2-093FR and DMR99-IRB-010-1). The IRB also specifically waived the consent requirement.Between January 2007 and December 2013, we retrospectively analyzed 53 patients with newly diagnosed oropharyngeal or hypopharyngeal squamous cell carcinoma who were ready to receive definitive chemoradiotherapy or radiotherapy for organ preservation at China Medical University Hospital. All patients had undergone pretreatment 9, gene expression profiles can be classified into hypoxic markers 10, radioresistant markers 11, a proliferative marker (Ki-67)12, a tumor progression factor (EGFR)13, and a surrogate marker for human papillomavirus 14. The spots of each pretreatment incisional biopsy were microscopically selected and arranged pairwise in tissue microarray blocks. Each tumor was represented by one tissue core on a tissue microarray. Furthermore, 4-µm-thick paraffin sections were deparaffinized and microwaved according to standard procedures before being processed for IHC staining.As described previously using IHC9, nuclear staining was performed for HIF-1α and Ki-67, whereas only cell membrane staining was performed for EGFR, CAIX, c-Met, Claudin-4, and Glut-1. VEGF and Bcl-2 exhibited a membranous or cytoplasmic staining pattern. YAP-1 was visualized through cytoplasmic or nuclear staining.Tissue slides were scored by two pathologists blinded to the study endpoints, and any disagreement between the two observers was resolved through consensus. This was because data dichotomization in IHC studies could distort the exact correlation between the study endpoints and protein biomarkers. Continuous scoring of the biomarkers using an H-score were reported, which was derived through a summation of the percentage of area stained at each intensity level multiplied by the weighted intensity. As detailed previously16. Representative images of IHC staining for PD-L1 are illustrated in Appendix 1.Tumor PD-L1 biomarker was evaluated through IHC staining using DAKO clone 22C3 pharmDx . Formalin-fixed paraffin embedded tumor tissues were deparaffinized and dehydrated in xylene and graded ethanol solutions. PD-L1 expressions were scored according to a tumor proportion score, which was defined as the percentage of tumor cells with complete or partial membranous staining at any intensityp16 overexpression detected through IHC staining was considered a surrogate marker for HPV involvement, which was verified in ten patients through the detection of HPV DNA using chromogenic in situ hybridization and polymerase chain reaction. Moreover, p16 expression was scored as positive when strong and diffuse nuclear and cytoplasmic staining was present in ≥70% of tumor cells14. Representative images of p16 positive cells are illustrated in Appendix 2. Fourteen patients (26%) were identified as having HPV-associated cancers. Tumors originated in the oropharynx and hypopharynx in 30 and 23 patients, respectively. The median age of the patients was 51 years. We performed tumor staging according to the American Joint Committee on Cancer criteria and observed that 7 and 46 patients had stage III and IVA-IVB cancer, respectively. Patient characteristics are listed in Table In this study, 18F)-FDG, and FDG PET/CT imaging was conducted approximately 60 minutes after the administration of 370 MBq of 18F-FDG. Thus, FDG uptake was determined in order to calculate the standardized uptake value (SUV). The maximum SUV (SUVmax) was confirmed through consensus between two nuclear medicine physicians.All patients were scanned using a PET/CT scanner . The patients were requested to fast for at least 4 hours before the administration of  of a tumor was delineated through an adaptive threshold method using a signal to background ratio. A voxel was defined as a local maximum if its SUV was not smaller than those of its neighbors. The SUV18, neighboring gray-level dependence matrix19, gray-level run length matrix (GLRLM)20, and gray-level size zone matrix (GLSZM)021. Because the definitions of GLCM and GLRLM are directional, 13 matrices were calculated for all possible orientation settings for each matrix. Finally, the textural features defined for each matrix were calculated. In total, 41 textural features or histograms were extracted (Appendix 3). The features were adopted on the basis of their ability to predict local recurrence after definitive radiotherapy or chemoradiotherapy, as previously described8.The heterogeneity of a tumor was evaluated using its textural features. The SUVs within a tumor were discretized using fixed bin widths of 0.05, 0.1, 0.2, 0.25, and 0.5 g/mL. For each discretization, four matrices were calculated to describe the texture of SUVs within the tumor: the gray-level co-occurrence matrix (GLCM)p < 0.05 was considered statistically significant. For statistically significant textural features associated with PD-L1 expression, we determined the optimal cutoff by the couple sensibility–specificity using ROC analysis. Statistical analyses were performed using SPSS, version 16.0 .Correlations between PD-L1 expression and different IHC studies or textural features were examined using Spearman’s rank correlation coefficient, with the alpha level set at 0.01. To compare their predictive ability for PD-L1 expression, all of the extracted features were first examined through receiver-operating characteristic (ROC) curve analysis using the aforementioned discretization methods. The abilities to predict mutational status were compared through examining the area under the curve (AUC). The optimal discretization method for each feature was chosen and entered for further analysis. If the AUC for PD-L1 expression was statistically significant, the quantitative differences for these indices or IHC studies between tumors with and without PD-L1 expression were examined using a Mann–Whitney U test. Thereafter, all of the statistically significant textural indices combined with IHC parameters were tested with logistic regression analysis to seek the independent predictable factors for PD-L1 expression. All analyses were two-sided, and All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.The IRB also specifically waived the consent requirement.The four groups of textural indices, the conventional PET-related parameters, the histograms, and the IHC staining intensities of the biomarkers were all retrieved for the entire cohort. The mean percentage of tumor PD-L1 expression was scored . With different cutoffs, 12, 18, and 30 tumors had PD-L1 expressions of ≥10%, ≥5%, and ≥1%, respectively.Ki-67 , c-Met , and p16 . The three IHC biomarkers were not related to each other . With p16 expression ≥70% as a surrogate of HPV infection, the proportion of tumors having PD-L1 expressions of ≥5% and ≥1% was 50% and 71% in patients with HPV-positive tumors (N = 14) and 28% and 51% in those with HPV-negative tumors (N = 39).The PD-L1 expressions were positively correlated with p = 0.04, γ = −0.27), run percentage , and short-zone low gray-level emphasis .For the various textural features, PD-L1 expression intensity was inversely correlated with gray-level nonuniformity for run ; short-run emphasis (SRE), long-run emphasis (LRE), run length nonuniformity (RLNU), GLNUr, and RP from GLRLM (26-connected and bin width = 0.5); coarseness and contrast from NGLDM (18-connected and bin width = 0.2); and gray-level nonuniformity for zone (GLNUz) and zone length nonuniformity (ZLNU) from GLSZM (18-connected and bin width = 0.5). When dichotomizing the PD-L1 expression levels at 1%, the ROC curves revealed that the good predictive performance of Ki-67 and p16. However, SZLGE became a sole feature for PD-L1 expression among the indices.Because few tumors had PD-L1 expressions of ≥10%, a binary classification of PD-L1 expression using this cutoff was excluded from the analysis. Table Ki-67, p16, and 12 textural indices were predictive for PD-L1 expressions of ≥5%. The logistic regression analysis indicated that the p16 staining percentage and correlation from GLCM were two independent predictors for PD-L1 expression. The odds ratios (ORs) were 17.00  and 0.009 , respectively. Figure p16 and correlation from GLCM between tumors with PD-L1 expressions of ≥5% and < 5%, as well as the corresponding ROC curves. The mean percentage intensity of p16 for tumors with PD-L1 expressions of ≥5% and < 5% were 50.28 ± 36.56% and 25.91 ± 27.38%, whereas the values of correlation from GLCM were −0.04 ± 0.45 and 0.31 ± 0.24 for the two groups, respectively.The quantitative differences are listed in Table p16 and Ki-67 staining percentages were two predictors for PD-L1 expression. The ORs were 11.41  and 757.77 , respectively. The quantitative difference of the two biomarkers between tumors with PD-L1 expression of ≥1% and < 1% are illustrated in Fig. When dichotomizing PD-L1 expression at 1%, the logistic regression analysis revealed that the p16 of 15% was chosen, the sensitivity, specificity, and accuracy for predicting PD-L1 expression of ≥5% were 78%, 46%, and 57%, respectively. If the cutoff for PD-L1 expression was 1%, the corresponding values were 77%, 57%, and 68%, respectively.Based on the aforementioned predictive parameters, we attempted to determine the optimal cutoffs for the biomarkers to distinguish tumors above and below the cutoffs of PD-L1 expression. When a cutoff value for %, the sensitivity, specificity, and accuracy of predicting PD-L1 expression of < 5% were 66%, 67%, and 66%, respectively.Moreover, correlation from GLCM was a negative predictor for PD-L1 expression of ≥5% (AUC = 0.24). When using an optimal cutoff of 0.26YAP1 and EGFR, correlation was observed between the studied biomarkers and certain textural features (Appendix 4). Several textural features from GLCM and NGLDM were correlated well with Claudin-4 levels, and others were related to hypoxic markers such as VEGF or HIF-1α. Appendix 5 shows the quantitative values of textural indices that were associated with the intensity of biomarkers according to the 50th percentile of the IHC score, or the aforementioned optimal cutoffs9. In summary, the overexpression of hypoxic markers  was positively associated with the values of entropy (GLCM), GLNUz, and RLNU. In addition, high gray-level run emphasis (HGRE), short-run high gray-level emphasis (SRHGE), high gray-level zone emphasis (HGZE), and short-zone high gray-level emphasis (SZHGE) were all positively related to VEGF, Glut1, and Ki-67 levels, but negatively related to Claudin-4 and c-Met. Furthermore, contrast (GLCM) and dissimilarity were positively associated with VEGF and Glut1 levels, but negatively related to Claudin-4 and c-Met. When using a cutoff of 30% for CAIX expression, homogeneity and variance were related to CAIX level.Except for 22. Although PD-L1 expression in numerous types of tumor has increased the chance of clinical outcomes in some cancers with anti-PD-1–directed therapy, the lack of a clear definition of positive tumor PD-L1 staining through IHC is problematic. Cutoffs for a positive result range from > 1% to > 50% based on the percentage of tumor cells stained, which suggests a connection with the existing issue of PD-L1 expression heterogeneity within the microenvironment5. By integrating several protein biomarkers and FDG-PET-based textural features, this pilot study revealed the association between tumor PD-L1 expression and tumor microenvironments or radiomics. Although IHC study for PD-L1 expression remains a standard care option, imaging studies or adjunctive biomarkers can provide additional information to circumvent the dynamic nature of immune microenvironments, or when timely biopsy cannot be employed.Immunotherapy represents a paradigm shift in cancer treatment; checkpoint inhibitors targeting the PD-1/PD-L1 axis have been reported to provide promising clinical responses in patients with various types of cancer23, with a positive detection rate of 20–35% obtained through IHC with a 5% cutoff. In addition, the PD-L1 expression according to HPV status was also in agreement with that of a molecular study24, which reported that 70% of HPV-associated and 29% of non-HPV-associated HNCs had PD-L1 expression at the same cutoff. Theoretically, PD-L1 is biologically active only when expressed on the cell membrane, either through dynamic IFN-γ expression or constitutive oncogene activation25. Oncogene-driven PD-L1 expression is characterized by the lack of an immune infiltrate4. Several studies have demonstrated the associated pathways related to oncogene activation, including PTEN loss27, EGFR activation28, and tumor hypoxia29. By contrast, a positive link between tumor p16 and PD-L1 expression in our study highlighted the model through which IFN-γ and other cytokines associated with an immune response can induce PD-L1 in tumor cells24. Therefore, in patients with HPV-associated HNC, our data (as well as two other studies) suggest a rationale for the therapeutic blockade of the PD-1/PD-L1 pathway24. Moreover, two additional studies have investigated the prognostic role of PD-L1 and other clinicopathologic features in patients with breast cancer31 and revealed that tumor PD-L1 expression is associated with an increased proliferation index of Ki-67. These findings were also observed in our data using the HNC cohort, although few biological studies have explored the molecular mechanism linking Ki-67 and the PD-L1 expression pathway. Therefore, more studies across various cancers are required to clarify the biological process.The ratio of tumor cell PD-L1 expression in our cohort was compatible with other HNC clinical trialset al.6 conducted a pilot study to examine the associations between FDG-PET and the immune-active features of the tumor microenvironment. They found significant correlations between SUVmax and SUVmean and the expressions of CD8-TILs and PD-1-TILs. However, no close relationship existed between the metabolic parameters and tumor cell PD-L1 expression. By using comprehensive FDG-PET-related textural features, the current study was the first to indicate that metabolic imaging phenotypes are able to predict tumor PD-L1 expression. To maximize predictive accuracy, a large sample size combined with machine learning processes for the selection of the discretization method may facilitate optimization of the performance of the textural features. If our findings are reproducible in additional validation studies, metabolic imaging features can be implemented to provide additional information if biopsies cannot be performed.Lopci 32. Although one study reported that there is an association between SUVmax of FDG and 18F-labeled nitroimidazoles (FMISO) in head and neck tumors33, future prospective studies are required to compare FMISO and textural features of FDG to image the tumor hypoxia. Currently, little evidence exists to support a straightforward correlation between textural heterogeneity and any specific underlying physiological processes or biological heterogeneity, our findings suggest that future studies could clarify the molecular mechanisms that may be related to the interplay between imaging phenotypes and tumor microenvironments.In addition, this study was the first to suggest that metabolic textural features from FDG-PET can indicate characteristics of some tumor microenvironments including hypoxia, angiogenesis, radioresistance, and tumor proliferation. To date, because no robust evidence exists to definitively dichotomize these protein biomarkers by differentiating treatment outcomes, we categorized them according to the 50th percentile or the optimal cutoffs reported in our previous study. In particular, several textural features were associated with tumor hypoxic markers. In oncology, identifying intratumoral areas with hypoxia is crucial because several studies on HNCs have concluded that partial oxygen pressure is one of the strongest independent predictors of overall survival, regardless of the treatment modality4. To elucidate the cross-talk between various immune parameters or cells, the use of larger tissue specimens instead of paraffin embedded tumor tissues is essential to recruit more immune-active markers. Furthermore, our study could not imply the correlation of FDG-PET radiomics or IHC biomarkers and the therapeutic effects with anti-PD-1 or anti-PD-L1 antibodies since the PD-L1 expression does not entirely associate with the therapeutic effects. Therefore, textural features in 18F-FDG-PET should be investigated as potential biomarkers for checkpoint inhibitors. Finally, features derived from FDG-PET-CT or IHC biomarkers remain insufficient to replace IHC testing for PD-L1 because predictive specificity and accuracy were not completely acceptable. To maximize their supplemental roles, a combination with various features or more biomarkers should be tested as a potential approach. Nonetheless, our results provide an initial step to link imaging phenotypes or IHC biomarkers and immune-active landscapes in order to maximize therapeutic strategies for checkpoint inhibitors.The findings of this study should be interpreted cautiously because of the small sample size and retrospective study design. External validation studies using an independent dataset with similar imaging and IHC studies are necessary to confirm these findings. The results would have been more robust if more immune features from tumor microenvironments, such as CD8-TILs, PD-1-TILs, and CD68 tumor-associated macrophages, were included under the scope of our analysis. However, when tumor PD-L1 expression was observed, it was frequently associated with infiltrating immune cellsp16 and Ki-67 staining percentages detected using IHC and several 18F-FDG PET/CT-derived textural features can provide supplemental information to determine tumor PD-L1 expression. The PD-L1 expressions were positively correlated with p16 and Ki-67, whereas the textural index of correlation was a negative predictor for PD-L1 expression of ≥5%. Further studies are required to validate our findings and to maximize the predictive accuracy.In patients with HNC, Appendix


The precursor was calcined in N2 flow under atmospheric pressure and thereafter, a humidity sensor based on the composite was fabricated. The sensor showed high resistive sensitivity and varied by more than four orders of magnitude with an increase in relative humidity (RH) from 11% to 95%. The response and recovery time were about 3.1 s and 76 s, respectively with maximum hysteresis at 1% RH. In comparison with pure SrTiO3 and black TiO2, the gray composite based device exhibits a higher sensitivity. These results demonstrate the potential of gray TiO2/SrTiO3 for humidity sensing applications.A gray TiO In 1996ications ,9.3 is recognized as one of the most promising perovskite oxides for sensing applications. Its wide band gap and large exciton binding energy induce excellent electrical and optoelectronic properties [3 based sensors and the primary focus has been on the conventional white SrTiO3 .SrTiOoperties , owing t3 such as humidity sensing, gas sensing, or photo-sensing characteristics have never been reported. Some research work regarding dark colored semiconductors was published by X. Chen in 2011. The black TiO2 with a narrow band-gap of 2.18 eV shows great solar absorption for photocatalysis application [2 has attracted considerable attention due to their enhanced photoelectric properties [2/SrTiO3 composite with a narrow band-gap was successfully prepared by a facile one-step synthetic method. The gray composite exhibits better humidity sensing performance in comparison with pure SrTiO3. Moreover, a narrow band-gap would be more favorable for generation of excited carriers and an increase in conductivity. Hence, sensors based on the dark colored semiconductors might possess more flexibility in realization of high performance multifunctional sensors, which endows composites with wider applications. However, to the best of our knowledge, the sensing properties of dark colored SrTiOoperties . In the 2/SrTiO3 composite nanocrystalline was synthesized in order to be used as a sensing material. Thereafter, the humidity sensor was fabricated by coating the material on carbon interdigitated electrodes. The humidity sensing characteristics were studied by measuring the impedance at different relative humidity (RH). The device shows excellent sensitivity, good linearity, and small hysteresis, which demonstrates that gray TiO2/SrTiO3 composite is a suitable candidate for humidity sensors.In the current work, gray TiO3)3 (Sr:Ti = 1:1) was added into the mixture under magnetical stirring. The resulting material was calcined in N2 flow under atmospheric pressure at 500–800 °C. The material synthesized at low temperature was TiO2/SrTiO3 composite; while that obtained at high temperature was more apt to form pure SrTiO3. The synthesized sample was mixed with deionized water in a weight ratio of 5:1 to form a paste. The paste was coated on a ceramic substrate (1.0 cm × 0.5 cm) with four pairs of carbon interdigitated electrodes to form a sensitive film. The film was dried in vacuum at 60 °C. The preparation details of black TiO2 are given elsewhere [In the experiment, 14 mL tetrabutyl titanate was dissolved in 20 mL ethanol. Then, 1 g urea (used for N doping) was dropped to obtain solution A, whereas solution B contained 5 mL deionized water, 10 mL ethanol, and 1 mL hydrochloric acid. Thereafter, solution B was gradually added dropwise to solution A under stirring with a glass rod until a white colloid was formed. Thereafter, colloid was kept in a water bath heated at 35 °C for half an hour. Subsequently, equimolar Sr2, NaCl, KCl, and KNO3 and the corresponding RH values were 11%, 33%, 54%, 75%, 85%, and 95%, respectively. The voltage was set at AC 1 V and the frequency changed from 40 Hz to 100 kHz in humidity studies. All the above device performance tests were operated at room temperature controlled by air conditioning.The crystal structure of the sample was examined by X-ray diffraction . The optoelectronic characterization of the films was examined by PerkinElmer UV/Vis spectrometer  at room temperature while Scanning electron microscope (SEM)  was used to investigate the morphology. The electrical properties of the sensor were measured by a Precision Impedance Analyzer 6500B Serious from Wayne Kerr Electronics . For the humidity sensing properties test, the sensor was put into six chambers with different RH values. The RH values ranging from 11 to 95% were obtained via using different saturated salt solutions as humidity generation sources. These saturated salt solutions were LiCl, MgCl2, gray TiO2/SrTiO3 composite and pure SrTiO3 are shown in 3 and brookite TiO2 composite. The black sample corresponds to the black TiO2 with anatase type, and the third sample shows pure SrTiO3 with perovskite structure.The X-ray diffraction (XRD) patterns of black TiO2/SrTiO3 composite is shown in 3. According to the relationship between band-gap and absorption edge, Eg = 1240/λ , the band gap of composite is estimated to be less than 2 eV [The dark color indicates the enhanced light absorption of the sample. The UV-visible absorption spectrum of gray TiOhan 2 eV . These r2/SrTiO3 composite studied via SEM at different resolutions. It shows that the composite has a cube shape and the average size is about 150 nm. A uniform and crack-free TiO2/SrTiO3 sample is obtained, demonstrating that the composite is suitable to act as a substrate material for humidity sensors.2/SrTiO3 humidity sensor at various frequencies is shown in 3-structured semiconductors reported previously, such as SrTiO3 nanospheres, NaTaO3, BaTiO3 [The relationship between impedance and RH for TiO, BaTiO3 ,8,14. Th, BaTiO3 .Additionally, at low frequencies, RH has a close-to-linear relationship with impedance, while there is no clear linearity of relationship at high frequencies. A high sensitivity and good linearity can be obtained at 100 Hz. Hence, 100 Hz was chosen as the ideal working frequency. Moreover, the impedance of referent ceramic substrate remained constant w.r.t change in RH, which signifies that the substrate has no contribution in humidity sensing.3 device changes by about three orders of magnitude while that of black TiO2 changes within one order of magnitude. The gray sample-based sensor exhibited a much higher sensitivity in comparison with that of black TiO2 and pure SrTiO3. The composites composed of different materials usually combine advantages of the components with better properties. For example, In2O3-NiO, ZnO-In2O3 and MoO3-NiO composites all exhibited enhanced humidity sensitive characteristics [eristics ,15,16. T2/SrTiO3 sensor appeared to have a narrow hysteresis loop. The maximum hysteresis of the device was about 1% RH, which was smaller than that of the pure SrTiO3 (less than 2% RH as measured).The response time is the time taken by a sensor to achieve 90% of the total resistance change in the case of adsorption process or the recovery time in the case of desorption process. Since the humidity detection range primarily covers the range of 11% RH–75% RH, the response and recovery characteristics of the sensor in this range were measured at 100 Hz, as shown in the inset. The response time for the change from 11% RH to 75% RH was about 3.1 s, and recovery time for the change from 75% RH to 11% RH was about 76 s.2/SrTiO3 sensor at different RH are presented in 3O+) [3O+ is low and the transfer of carriers is difficult, the charge carriers would bring about electrons accumulation and bend the energy-band at the grain surface, leading to a decreased resistance. Meanwhile, bounded electrons emerge by means of polarization of the sensing material. At this time, the dominant conducting carriers are H3O+ and bounded electrons [To have an enhanced understanding of the sensing mechanism, the complex impedance plots of TiO in 3O+) . Though lectrons .Starting from the spectrum of 54% RH, an evident straight line in the low frequency region appears which is the consequence of diffusion and polarization of charged carriers between sensing film and electrodes . With th3 increases as the frequency increases, then the polarization of adsorbed water molecules is enhanced with increasing RH [It is well known that the characteristic frequency corresponds to the value when the imaginary part of the complex impedance spectrum reaches its maximum, near which is known as the dispersion region. Seen from the spectra in asing RH .2/SrTiO3 were investigated. The impedance changes by more than four orders of magnitude in the range from 11% to 95% RH at 100 Hz, and the maximum hysteresis is about 1% RH. Furthermore, the sensing mechanism was discussed by the complex impedance spectra in detail. These results demonstrate the potential application of the composite in fabricating high performance humidity sensors.In conclusion, the synthesis, characterization, and humidity sensing of TiO


When thinking about RNA three-dimensional structures, coming across GNRA and UNCG tetraloops is perceived as a boon since their folds have been extensively described. Nevertheless, analyzing loop conformations within RNA and RNP structures led us to uncover several instances of GNRA and UNCG loops that do not fold as expected. We noticed that when a GNRA does not assume its “natural” fold, it adopts the one we typically associate with a UNCG sequence. The same folding interconversion may occur for loops with UNCG sequences, for instance within tRNA anticodon loops. Hence, we show that some structured tetranucleotide sequences starting with G or U can adopt either of these folds. The underlying structural basis that defines these two fold types is the mutually exclusive stacking of a backbone oxygen on either the first (in GNRA) or the last nucleobase (in UNCG), generating an oxygen–π contact. We thereby propose to refrain from using sequences to distinguish between loop conformations. Instead, we suggest using descriptors such as U-turn (for “GNRA-type” folds) and a newly described Z-turn (for “UNCG-type” folds). Because tetraloops adopt for the largest part only two (inter)convertible turns, we are better able to interpret from a structural perspective loop interchangeability occurring in ribosomes and viral RNA. In this respect, we propose a general view on the inclination for a given sequence to adopt (or not) a specific fold. We also suggest how long-noncoding RNAs may adopt discrete but transient structures, which are therefore hard to predict. RNA architecture is modular and hierarchical, which implies that secondary structural elements such as double stranded helices, hairpins, and single-stranded loops are linked by tertiary interactions that guide the assembly process . The majHere, we present structural evidence that challenges these expectations by identifying GNRA sequences that adopt a UNCG fold and vice versa, both in tetraloops closed by a Watson–Crick base pair and in tetraloop-like motifs embedded in larger ribosomal and tRNA loops . AlthougTo better characterize these interconversions, we propose a more general structure-based tetraloop and tetraloop-like identification scheme that involves on one side the classical and well-described U-turn  and, on +UAAu loop (+ imino or amino nitrogen atom—and an OP atom of the fourth nucleotide. This base–phosphate hydrogen bond is of the “5/4/3BPh” type according to a recent classification (trans-Sugar/Watson–Crick pair (t–SW) occurring in GNRA loops should not be considered as a U-turn determinant although it is essential for interactions with GNRA receptors  hydrogen bond imposes the formation of an oxygen–π or phosphate–π stacking contact between the first nucleobase and an OP atom of the third nucleotide. A PDB survey led to an average OP–π stacking distance of 3.0 ± 0.2 Å, with a maximum distance of 3.5 Å. This oxygen–π contact, which is a further characteristic of U-turns, has rarely been described .It emerges that these two features, namely the 1–4 nucleobase–OP hydrogen bond and the OP–π stacking contacts, are sufficient to unambiguously characterize a U-turn. The latter criterion allows us also to distinguish between regular and partially degenerated or unfolded U-turns, which correspond to loops with no oxygen–π stacking contact and are most often found at RNA–protein interfaces. However, such occurrences are rare (see the following section).trans-Sugar/Hoogsteen (t-SH) pair instead of a hydrogen bond involving the OP atom of the fourth nucleotide as in more typical U-turns. Yet, in the examples we collected, the OP–π contact between the first nucleobase and an OP atom of the third nucleotide is conserved. In the following, we call this U-turn variant a “USH-turn” because of the consistent presence of a 1–4 t–SH pair.A U-turn variant has been identified for UNAC sequences B. These t–SW pair  interaction between the first and fourth nucleobase, associated with a C2′-endo pucker of the third residue, and a syn conformation of the fourth residue. In addition, the third and fourth ribose rings adopt an uncommon head-to-tail orientation  pair. As such, these Zanti-turns are also known as di-loops. Interestingly, the characteristic C2′-endo sugar pucker of UNCG tetraloops seems to be conserved in all Z-turn types.Such a definition is not based on the UG folds . Hence, + are the most frequent, followed by Z-turns in UNCG tetraloops. USH-turns are less frequent and are associated with UNAC sequences. Zanti-turns are slightly more frequent and diverse and comprise essentially CNNG sequences. The “Uncategorized” motifs are mostly of the partially unfolded U-turn type—where the 1–4 interaction is present, but not the OP–π stacking contact. They correspond also to folds that are too rare and/or disordered to allow for their assignment to any clearly defined category, or to partially unfolded conformations induced by proteins. The rare GANC tetranucleotide loop has only been identified in group IIC introns based on structural and phylogenetic evidence and has only been reported when bound to its cognate receptor . These examples are more thoroughly described in the following sections. A detailed report describing the structural features of tetranucleotide folds will be provided elsewhere, the main purpose of this account being to establish the interchangeability between U-turns and Z-turns.However, these data also indicate that some sequences expected to form a U-turn are associated with a Z-turn and vice versa. Thus, the sequence of a tetraloop does not systematically dictate its fold. For instance, we identified a GCAAu sequence that adopts a Znti-turn . Furthernti-turn A. NMR stt–SW pair   —adopts a: 3.4 Å) B. Althouturn see A, 2.t-SH pair  is capped either by a CUUG (45%), a UUCG (36%), or a GCAA (13%) tetraloop  might interrogate classical phylogenetic data interpretations. Indeed, in the seven UNCG tetranucleotide sequences deduced from the 16S Escherichia coli 2D structure, only three adopt a canonical Z-turn and the other sequences appear in disordered regions with, however, a G nucleotide in syn for four of them. The reasons as to why these loops appear as disordered are not yet understood.Phylogenetic data on 16S rRNA suggested early on that helix 6  histograms were calculated based on all oxygen–π contacts identified in RNA structures from the PDB and, therefore, not only on those found in tetraloop folds. To check for tetranucleotides with 1–4 interactions in NMR structures, we used the RNA FRABASE 2.0 database  for tetranucleotide sequences in RNA hairpins that involve a 1–4 nucleobase–nucleotide interaction and an oxygen-π contact as defined below. For that purpose, we used the DSSR program . DSSR wadatabase .syn/anti conformations, they were considered as similar and the one with the best resolution was labeled as nonredundant. In cases of matching resolutions, the nucleotide sequence with the lowest average B-factor was selected. Alike, if in a same structure two sequences shared the same residue numbers (with different chain codes) as well as ribose puckers, backbone dihedral angle sequences, and syn/anti conformations, they were considered as similar and the one corresponding to the first biological unit was marked as nonredundant. To further limit redundancy in the largest ribosomal structures, we restricted our analysis to a single biological assembly. For more details, see For


Self-splicing introns populate several highly conserved protein-coding genes in fungal and plant mitochondria. In fungi, many of these introns have... Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Lachancea kluyveri, we expose nonrandom patterns of genetic diversity in exons that border self-splicing introns. In particular, we show that, in all three species, the density of single nucleotide polymorphisms increases as one approaches a mobile intron. Through multiple lines of evidence, we rule out relaxed purifying selection as the cause of uneven nucleotide diversity. Instead, our findings implicate intron mobility as a direct driver of host gene diversity. We discuss two mechanistic scenarios that are consistent with the data: either endonuclease activity and subsequent error-prone repair have left a mutational footprint on the insertion environment of mobile introns or nonrandom patterns of genetic diversity are caused by exonic coconversion, which occurs when introns spread to empty target sites via homologous recombination. Importantly, however, we show that exonic coconversion can only explain diversity gradients near intron–exon boundaries if the conversion template comes from outside the population. In other words, there must be pervasive and ongoing horizontal gene transfer of self-splicing introns into extant fungal populations.Self-splicing introns populate several highly conserved protein-coding genes in fungal and plant mitochondria. In fungi, many of these introns have retained their ability to spread to intron-free target sites, often assisted by intron-encoded endonucleases that initiate the homing process. Here, leveraging population genomic data from  Saccharomyces cerevisiae , often singular target motif with high affinity (SELF-splicing introns are selfish elements with a broad but patchy phylogenetic distribution. Found in transfer RNA (tRNA), ribosomal RNA (rRNA) and  protein-coding genes in bacteria and archaea, they are particularly numerous in mitochondrial genomes of fungi and plants where they have invaded genes encoding components of the electron transport chain (ETC) . In manyaffinity , accompaMAT) locus of S. cerevisiae, which is cleaved by the endonuclease HO and subsequently repaired via HR  patterns in exons flanking mitochondrial self-splicing introns.We reasoned that one way to develop a greater understanding of such on-target mutagenicity would be to study endonucleases in their native genomic context. If endonuclease activity is indeed mutagenic, cleavage and repair might have left a detectable imprint on population-wide genetic variation around the cleavage site. In search of such an imprint, we survey recent high-quality population genomic data from S. cerevisiae mitochondrial genomes, originating from a recent high-coverage resequencing effort  with the terminal exons of cob and cox1 from the reference S288C genome, we identified unique full-length cob and cox1 genes in all strains, capturing both coding exons and intervening introns. We aligned the 94 sequences (93 plus the S288C reference genome) for each gene together with 50 nt of upstream/downstream flanking DNA using MUSCLE v. 3.8.31 , respectively. Identification and alignment of the MATα  and Z1 regions based on the S288C annotation were straightforward, given the exceptional conservation levels reported below. Genes containing nuclear spliceosomal introns in S228C were identified based on GenBank annotations. The terminal exons of these genes (required minimum length >1 nt) were then blasted against the remaining genomes . Homologous sequences were extracted for cases where both terminal exons were at least 70 nt long, identified as the only hits in the BLAST query, located on the same chromosome and strand, and <3 kb apart . Homologous sequences recovered in at least 80 strains were then aligned using MUSCLE with default parameters.For the analysis of the cox1 and cob coding sequences for 18 L. kluyveri strains from Paul Jung (University of Luxembourg). Intron positions in these strains were taken from supplemental material, figure S2 of S. cerevisiae) and subsequent manual inspection, we conservatively excluded strains CBS10367 and CBS10368 because of conspicuous divergence at the 5′ end of exon 4.We obtained L. kluyveri strains by In inferring distances to the nearest intron–exon boundary, we considered all intron insertion sites observed across the 18 S. pombe strains (https://danieljeffares.com/data/). In the absence of high-quality de novo mitochondrial assemblies for these strains and unknown intron presence/absence variability, we only considered introns present in the S. pombe reference genome when inferring distances to the nearest intron–exon boundary.We obtained variant calls across 161 Arabidopsis thaliana chloroplast and mitochondrial genomes were obtained from the 1001 Genomes Project  to assess the degree to which polymorphic exonic sites were involved in pairing to intronic residues as part of the self-splicing process. Overlap with endonuclease cleavage sites was assessed based on cleavage motifs defined in REBASE v608 (S. cerevisiae introns were defined as in S. cerevisiae and L. kluyveri and the standard code (NCBI transl_table = 1) for S. pombe.We use structural information provided by the Group I Intron Sequence and Structure Database (GISSD) , we first considered SNP density as a function of distance from the nearest intron–exon boundary across cox1 and cob exons. SNP density here is defined as the number of SNPs at a given distance (≤70 nt) from the nearest intron boundary, divided by the number of exons that contribute a nucleotide at that distance . We observe a marked increase in exonic SNP density as one approaches the intron–exon boundary .The and cox1 . WhereasP = 0.04) and group II introns  and is broadly similar for 5′ and 3′ exon ends, with a marginally smaller contribution of 3′ exon ends . This might be linked to a greater fraction of nucleotides at 3′ exon termini being under selection to maintain splice-relevant base-pairing interactions with the neighboring intron (see below). Inevitably given the area of SNP enrichment, a substantial proportion of boundary-proximal polymorphisms are located in known endonuclease cleavage motifs . This might be considered surprising. However, systematic mutagenesis experiments previously demonstrated that many single-nucleotide changes do not perturb target recognition and cleavage (cox1) had all been tested individually and found to have wild-type cutting efficacies . Exon ends bordering immobile mitochondrial introns do not show a similar enrichment for SNPs . Similarly, we find no SNP enrichment in the exonic borders of spliceosomal nuclear introns, which lack the capacity to excise themselves from their host messenger RNA (mRNA) and do not encode endonucleases or other mobility factors , whose mitochondrial and chloroplast genomes also harbor self-splicing introns embedded in protein-coding genes. However, unlike their fungal counterparts, these introns lack open reading frames that encode functional endonuclease, RT, or other domains that might mediate mobility and, like those of other land plants, are not mobile as a consequence  had postulated the presence of mutational hotspots near intron–exon boundaries but lacked quantitative support , precludsequence . As predS. cerevisiae, most mobile introns are located in cox1, with only a single intron in cob reported to be mobile in crossing experiments , whereas cob exons do not . To rule out gene-specific factors rather than mobility in the genesis of nucleotide diversity, we examined mitochondrial protein-coding genes from 161 S. pombe and 16 L. kluyveri strains . The S. pombe reference genome encodes two introns in cox1 and a single intron in cob. Importantly, the group II cob intron alone is known to be mobile  in L. kluyveri, whereas in S. pombe a negative SNP gradient is evident for cob  but not cox1 .In eriments   are nonsynonymous, a significant depletion compared to the mutational expectation of ∼2/3 (Fisher test P = 0.001). Similarly, only one out of eight SNPs (12.5%) in close vicinity of the cobS. pombe  intron is nonsynonymous . In both cases, lower levels of nonsynonymous diversity support the notion of strong ongoing protein-level selection at intron–exon boundaries. Interestingly, we find a relatively large number of nonsynonymous mutations in cox1S. cerevisiae  (15/44 = 34%). However, the ratio is similarly high (5/19 = 26%) further away from the boundary (Fisher test P = 0.77), arguing for a global rather than local, boundary-anchored relaxation of constraint. This observation is broadly consistent with prior evidence for reduced selection on mitochondria in the wake of the whole genome duplication (WGD) event . Disruption of proper intron–exon base pairing is anticipated to impair splicing, with deleterious consequences for the host since splicing is required to reconstitute functional cob/cox1 reading frames. Although splice-relevant exonic residues are firmly located in the zone of enriched SNP density, we find only a single SNP at a nucleotide position that is involved in intron–exon base pairing; and even this SNP, a synonymous change (CAC↔CAT) at the 5′ end of cox1 exon 5, likely preserves base pairing (G-C↔G-T). This observation supports the notion that we do not see higher SNP densities as the result of relaxed selection but rather despite persistent functional constraint. In fact, if excess variation reflects differential mutational input, we are likely to underestimate the true mutational gradient, given the added splicing-related constraints in the immediate vicinity of introns.Further testimony for ongoing purifying selection at the intron–exon boundary comes from scrutinizing exonic residues involved in base-pairing interactions with the neighboring intron . We reasoned that local constraints on protein function and structure should be very similar between the human and yeast orthologs and that, in considering polymorphisms found in the constitutively intronless human orthologs, we circumvent potential circularity in assessing the relationship between introns and local conservation. We therefore charted SNP density across human cox1 and cob as a function of distance from mock splice junctions, placed at orthologous positions in the respective gene . We find no evidence for locally relaxed selective constraint for either gene, regardless of whether we assume S. cerevisiae or L. kluyveri intron positions . Such a scenario is not necessarily unreasonable, given prior findings, notably in plants, of high rates of horizontal transfer of self-splicing introns  of yeast crosses, which were attributed to microhomology-mediated end joining. Further, studying HR, MAT locus but found it to be perfectly conserved across the 92 S. cerevisiae strains analyzed here. The first SNP was found 192 nt downstream of the HO cutting site. This high level of conservation is indicative of exceptionally strong nucleotide-level constraint and echoes previous observations of very slow divergence (>96% nucleotide identity) between otherwise well-diverged Saccharomyces spp. (MAT locus as a model to study mutagenic effects.An alternative to the HGT plus exonic coconversion model is that intron insertion sites constitute mutational hotspots, as previously suggested (HO-initiated DSB repair is mutagenic might be specifically reflective of HR following endonuclease-mediated cleavage rather than HR in general. Is it possible that the activity or presence of the endonuclease itself affects the repair process? There have been a number of recent reports that DNA-binding proteins, by associating with a lesion-containing target site, can prevent proper damage surveillance and repair, ultimately leading to a higher incidence of mutations (td intron of phage T4, where the intron-encoded I-TevI endonuclease, which cleaves distally to its binding site, remains preferentially associated with one of the free cleavage products, and thereby asymmetrically impedes resection (It is worth considering at this point whether the finding that If nonrandom patterns of genetic diversity are indeed mutational in origin, our findings have important implications for the cost of self-splicing introns, which have generally been considered as relatively cost-free, given their ability to efficiently remove themselves from their host genes . Our reshttp://www.genetics.org/content/early/2017/02/13/genetics.116.199059.supplemental.Supplemental material is available online at Click here for additional data file.