Recent studies suggest that the incidence of idiopathic pulmonary fibrosis (IPF) is rising. Accurate epidemiological data on IPF, however, are sparse and the results of previous studies are contradictory. This study was undertaken to gain insight into the various methods used in the epidemiological research of IPF, and to get accurate and comparable data on these different methodologies.A systematic database search was performed in order to identify all epidemiological studies on IPF after the previous guidelines for diagnosis and treatment were published in 2000. Medline (via Pubmed), Science Sitation Index (via Web of Science) and Embase databases were searched for original epidemiological articles published in English in international peer-reviewed journals starting from 2001. After pre-screening and a full-text review, 13 articles were accepted for data abstraction.Three different methodologies of epidemiological studies were most commonly used, namely: 1) national registry databases, 2) questionnaire-based studies, and 3) analysis of the health care system’s own registry databases. The overall prevalence and incidence of IPF varied in these studies between 0.5–27.9/100,000 and 0.22–8.8/100,000, respectively. According to four studies the mortality and incidence of IPF are rising.We conclude that there are numerous ways to execute epidemiological research in the field of IPF. This review offers the possibility to compare the different methodologies that have been used, and this information could form a basis for future studies investigating the prevalence and incidence of IPF. Diagnostics and treatment of idiopathic pulmonary fibrosis (IPF) is currently undergoing changes. In the most recent guidelines , the diaEpidemiological data of IPF are sparse, and moreover, changes in classification of idiopathic interstitial pneumonias (IIP) and diagnostics make the comparison of the data from various decades difficult. Until the 1990s, idiopathic pulmonary fibrosis covered a heterogeneous group of different interstitial diseases. The ATS/ERS consensus statement of diagnosis and treatment in 2000  and clasNational IPF registries are being set up ,8, and wOur aim was to review the literature of IPF epidemiology that has been published after the previous international statement on IPF in 2000. Therefore, we performed a systematic literature search in September 2012 with the help of an information specialist at the University of Turku/Medical library. Relevant literature was identified through the databases Medline (via Pubmed) and Science Citation Index (via Web of Science) starting from 01/01/2001 onwards. Only articles published in English were included in the study. The search was limited to human adults, using the terms idiopathic pulmonary fibrosis, interstitial lung diseases and epidemiology, combined with geographical areas . The initial search resulted in 214 articles. Two authors  independently pre-screened titles and abstracts, and data extraction resulted in 28 articles. All articles containing demographic data, incidence, prevalence or mortality rates of IPF or interstitial lung diseases (ILD), also including IPF patients, were accepted.The 28 eligible articles were then full-text reviewed by four authors . By consensus, only international peer-reviewed original articles were included, leading to the exclusion of seven review articles and one comparative report of ILD registries in three European countries . Two stuAn additional search was conducted in June 2013 to complement the systematic analysis. Embase was searched using the same queries as initially. 942 articles were found and titles were pre-screened by one author (JK). One additional article – published after September 2012 – was added to this systematic review. A summary of the selection process of articles is presented in Figure After the final evaluation, 13 articles were selected for data abstraction Table .From the 13 articles chosen for this review, we identified three commonly-used methods for the evaluation of IPF epidemiology in a population, and used this knowledge to categorize epidemiological studies in order to simplify the comparison of heterogeneous population data (Table In Flanders—the northern part of Belgium—20 centers of respiratory medicine took part in the registration of ILD patients between the years 1992–1996 . The tweIPF was the second most common ILD 20%) after sarcoidosis. Of the 72 patients with IPF, 21 (29%) had biopsy-proven UIP, 10 patients had bronchiolitis obliterans organizing pneumonia (BOOP), nine had desquamative interstitial pneumonia (DIP), three had lymphocytic interstitial pneumonia (LIP) and in 29 cases (40%) the histology was not specified. The cases with biopsy proven IPF/UIP were identified according to the ATS/ERS 2000 guidelines . Again m0% after The Italian register for diffuse infiltrative lung disorders (RIPID) was established in 1998 . The meaBoth of these national registry studies rely considerably on the activity of physicians in reporting patient numbers and data, and studies performed in this way should always be interpreted knowing of this bias, that results in underestimation of patient numbers. Registry cohorts like these, are, however important in the determination of patient clinical course and the possible differences in the management of patients in different centers.Two methodologically similar articles were found from Greece and Spain, where a standardized questionnaire was sent to respiratory medicine centers in order to obtain information on patients’ clinical data, procedures used to establish the diagnosis and the exact diagnosis.6 inhabitants. A standardized questionnaire was sent to the centers. An ATS/ERS consensus statement from 2002 was applied for classifying ILDs. A total of 511 cases of ILD were registered. Idiopathic pulmonary fibrosis was the most frequent disease, covering 197 patients/38.6% of all ILD patients; thus, according to our calculation, this results in an incidence of 2.94/100,000. The diagnosis was established by a surgical lung biopsy in 31.5%, and clinical criteria including HRCT scanning in 68.5%.In Spain, a registry of incident cases of ILD was set up from October 2000 to September 2001 [In Greece, departments of Pneumonology were contacted, and pneumonologists were asked to complete a questionnaire for all ILD patients who were alive in 2004 , as well as for every new incident case diagnosed during 2004 . CentersDue to the low response rates and lack of monitoring of the centers that have attended the study, data derived from these two studies should be interpreted with caution.In Finland, the prevalence of sporadic and familial IPF was evaluated during the years 1997–1998 . All FinIn Norway, the incidence and prevalence of physician-diagnosed and hospitalised cryptogenic fibrosing alveolitis (CFA) was studied during 1984–1998 . Thus, tIn the United Kingdom, a computerized longitudinal general practice database  was analyzed to estimate the incidence of IPF and sarcoidosis between 1991–2003 . The terIn the United States, the annual incidence and prevalence of IPF was estimated by utilizing a large health care claims database from 01/1996–12/2000 . IPF patIn another study conducted in the United States, mortality to pulmonary fibrosis (PF) was studied from 1992–2003 . The NatClinical records were also utilized in Japan to gain epidemiological data of IIP . All IIPIn Olmsted County, Minnesota, during a nine-year period (01/1997–12/2005) residents were screened, and those with possible IPF were identified . These c—was also screened for diagnoses of IPF-CS between 2000–2008. Cases with a co-existing connective tissue disease, and individuals who had co-existing diagnoses of extrinsic allergic alveolitis, asbestosis, pneumoconiosis and sarcoidosis were excluded. The search resulted in a total number of 2,074 incident cases which equates to an incidence of 7.44/100,000. The majority (63%) of new cases were men. The mean age at diagnosis was 74.3 years. After adjusting for age, sex and health authority, the estimated annual increase in the incidence of IPF-CS was 5%. The overall median survival for this cohort was 3.03 years. The advantage in the methodology used here is the combination data derived from of two different and independent registries, but the results are handicapped with the lack of reevaluation of the diagnostic accuracy of these registries.In the United Kingdom, death registers and general practice databases were used to investigate mortality (1968–2008) and incidence (2000–2008) of IPF . The diaIn Taiwan, a large population-based study was conducted to collect epidemiological information of IPF among Asians . The datThe estimated prevalence, based on the broad case definition, ranged from 0.7 in 1998 to 6.4 in 2007. According to the narrow case definition the estimated prevalence was 0.5 in 1998 and 4.9 in 2007. The annual incidence was calculated to be 0.6 in 1998 to 1.4 in 2007 . The incidence ranged 0.5 in 1998 to 1.2 in 2007  or from IPF registries ,34. The In addition to the identification of epidemiological parameters, our primary aim was to identify plausible and reliable methods in studies related to IPF epidemiology, as the field of IPF medicine is undergoing considerable change due to specific drugs that are entering clinical use, and many nationwide registry databases which are currently being built. Various and very heterogeneous methods are being used by IPF researchers, and so far the data from these studies is not comparable between countries due to this variation. We identified three categories or strategies for collecting IPF epidemiological data: nationwide registries for IPF or ILD, such as the Italian registry; questionnaires for health-care professionals; and systematic searches from existing databases. From these methods, questionnaires seemed to produce the lowest prevalence rates compared to national registries and/or searches from pre-existing databases. The coverage of pre-existing databases might be high in cases such as those shown in the study of Navaratnam , but theThe number of incidental and prevalent cases varied greatly in the presented studies (prevalence from 0.5  to 27,9/Changes in diagnostic criteria may also explain some of the observed variation. Studies implemented before the ATS/ERS 2000 guidelines  have shoThe international disease classifications have also reflected the lack of a clear, systematic classification of ILD:s: only in the latest version of ICD-10 from 2013, there has been a separarate subclassification for IPF. It is to be expected, that studies performed after this disease classification has been taken into use, and in countries where the population registries are most reliable, will yield high-quality data on IPF epidemiology. There are already preliminary reports on several national registry studies, which hopefully will yield comparable data on IPF epidemiology in different geographical and cultural areas ,40,41.Overall, it can be concluded that the existing databases present an overestimation of patient numbers, whereas registries and questionnaire-type studies result in an underestimation of patient numbers. The acquirement of accurate epidemiological data is crucial in elucidating the natural course and prognosis of IPF in different populations, and can lead to a better understanding of the disease etiology. However, if epidemiological data are collected, it should include a broad systematic screening of registries in the health care system, and a specific reevaluation of the patient records, especially the evaluations of pulmonary pathologists and radiologists. Novel electronic databases will hopefully offer more comparable data in this setting in the near future.BOOP: Bronchiolitis obliterans organizing pneumonia; DIP: Desquamative interstitial pneumonia; HRCT: High resolution computed tomography; IIP: Idiopathic interstitial pneumonias; ILD: Interstitial lung diseases; IPF: Idiopathic pulmonary fibrosis; LIP: Lymphocytic interstitial pneumonia; NSIP: Nonspecific interstitial pneumonia; PF: Pulmonary fibrosis; UIP: Usual interstitial pneumonia; VATS: Video-assisted thoracic surgery.None of the authors have competing interests related to this work., revised the manuscript, RK: presented the original idea for this work, reviewed the original articles included in this work and participated in writing the manuscript, MM; reviewed the original articles included in this work, and contributed in the writing of the manuscript. All authors read and approved the final manuscript.JK: reviewed the original articles included in this work, collected data from these articles, and drafted and wrote the manuscript; E-RS: reviewed the original articles included in this work, and wrote the manuscript, UH:The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/53/prepub


Non-covalent interactions of aromatics are important in a wide range of chemical and biological applications. The past two decades have seen numerous reports of arene-arene binding being understood in terms Hammett substituent constants, and similar analyses have recently been extended to cation-arene and anion-arene binding. It is not immediately clear why electrostatic Hammett parameters should work so well in predicting the binding for all three interactions, given that different intermolecular forces dominate each interaction. This review explores such anomalies, and summarizes how Hammett substituent constants have been employed to understand the non-covalent binding in arene-arene, cation-arene and anion-arene interactions. Non-covalent interactions of aromatic π-electron density have been extensively studied . There imeta to the carboxylic acid functional group are termed σm, and are generally recognized as describing the movement of electrons via the σ-framework (inductive effects). The σp Hammett constant is obtained from substitution para to the –CO2H group, and it describes the movement of electrons via the σ- and π-framework (inductive and resonance effects). Hammett constants were developed to help explain trends in the reactivity of meta- and para-substituted benzoic acid derivatives and related compounds. For instance, the electrophilicity of meta- and para-substituted benzoic esters, the nucleophilicity of meta- and para-substituted anilines, and the solvolysis of meta- and para-substituted benzyl halides . A . A ‡, ofsyn and anti 1,8-di-o-tolylnaphthalenes where each tolyl group was substituted para to the naphthyl ring (p), and this led to the conclusion that charge-transfer effects should be expected to minimally contribute to the overall aromatic interaction as compared to the electrostatic contribution . Not. Not1]. –-substituted benzene complexes  and electron-donating  groups. All complexes were defined with the anion in the plane of the aryl CH and outside of the periphery of the aromatic ring. As would be expected, the presence of electron-donating substituents decreased the binding energy and lengthened the anion-arene distance, and electron-withdrawing substituents increased the binding energy and shortened the anion-arene distance meta- or stituent , 44, 46,stituent . The facstituent , 35, 37 m or Σσm, while others show σp or Σσp to be best. Why would the inductive contributions of a substituent, as measured by σm or Σσm, sometimes be a better predictor of the binding energies than the combination of the inductive and resonance effects, as measured by σp or Σσp? Certainly one cannot turn off the resonance withdrawing/donating abilities of a substituent, so why should σm or Σσm ever outperform σp or Σσp? Even more perplexing are the reported correlations with Σ∣σm∣ [Total, which equals (Σσm + Σσp) [m∣ value the authors suggest the absolute value of ∣σm∣ contains information about the electronic and polarizability properties of a substituent [Total suggest it contains information about both induction and resonance effects, but why is induction counted twice: once in the σm value and again in the σp value [m∣ value [Of equal interest to discussions about what forces dominate the various interactions, and the role of Hammett constants in predicting the binding energies, is why some studies show the best correlations with σth Σ∣σm∣  or σTotam + Σσp) . In the stituent , but thiσp value ? Note thm∣ value . We are m, while for others it is σp, and for others still it is some manipulation of these parameters such as ∣σm∣ or (σm+σp). In some cases the differences between the cited studies can be attributed to the investigation of different substituted aromatics. Still, this is not always the case, and significant work remains to determine why Hammett constants have performed so well in predicting the binding energies of the interactions discussed here, and why sometimes σm gives the better correlations, while in other instances σp performs best. Finally, as noted by Hunter, Cockroft and coworkers, the importance of dispersion in gas-phase interactions may be due to the lack of a desolvation term [The correlation of Hammett substituent constants with arene-arene, cation-π and anion-π binding energies has allowed researchers to comment on the forces that govern the interactions; however, we believe these studies have produced as many questions as they have addressed. At a few places in this review we have noted that some studies observe the best correlations with σion term , 39, and


The membrane proximal external region (MPER) of HIV-1 is an important target of broadly cross-reactive mAbs. CAPRISA participant, CAP206, developed anti-MPER antibodies early that became cross-neutralizing at 18 months post-infection. This coincided with neutralization of the C4 HIV-2/HIV-1 chimera containing the W670 residue, suggesting changes in an antibody paratope may have resulted in the acquisition of breadth. A neutralizing mAb (CAP206-CH12) was isolated, providing an opportunity to determine which somatic hypermutations contribute to breadth.http://www.dulci.org). Batch transient transfections were used to generate recombinant antibodies. Neutralization breadth and potency was tested against autologous, Tier 2, and HIV-2/HIV-1 MPER chimeric viruses using the TZM-bl assay. Binding to MPER peptides was assessed by ELISA and surface plasmon resonance (SPR).The putative CAP206-CH12 reverted unmutated ancestor (RUA) was inferred using SoDA  where changes near the CDRs in CAP206-CH12 were reverted back to the germline sequence (11 in the heavy and 5 in the light chain). This CAP206-CH12_IP did not neutralize the C4 chimera suggesting that changes responsible for the affinity-matured CAP206-CH12 neutralizing capacity were among these 16 residues. Chimeric pairs of the light chain IP with the heavy chain of CAP206-CH12 or CAP206-CH12_RUA showed binding to MPER with differences in binding kinetics.The reduced binding and neutralizing activity of CAP206-CH12_RUA and CAP206-CH12_IP compared to CAP206-CH12 suggests a correlation between affinity maturation, neutralization breadth and potency. Ongoing work will assess the affinity maturation of CAP206-CH12 by determining moieties in the antibody paratope associated with effective epitope recognition and the effects of somatic mutations on the evolution of neutralization.


Phonons, which are collective excitations in a lattice of atoms or molecules, play a major role in determining various physical properties of condensed matter, such as thermal and electrical conductivities. In particular, phonons in graphene interact strongly with electrons; however, unlike in usual metals, these interactions between phonons and massless Dirac fermions appear to mirror the rather complicated physics of those between light and relativistic electrons. Therefore, a fundamental understanding of the underlying physics through systematic studies of phonon interactions and excitations in graphene is crucial for realising graphene-based devices. In this study, we demonstrate that the local phonon properties of graphene can be controlled at the nanoscale by tuning the interaction strength between graphene and an underlying Pt substrate. Using scanning probe methods, we determine that the reduced interaction due to embedded Ar atoms facilitates electron–phonon excitations, further influencing phonon-assisted inelastic electron tunnelling. A fundamental understanding of electron–phonon interactions in graphene is crucial to developing graphene-based electronic and photonic devices. Here, the authors control the properties of phonons in graphene by tuning the interaction strength between graphene and an underlying platinum substrate. Graphene, which is known to exhibit linear energy dispersion near the Fermi level, is considered a highly desirable material for future electronic1247910111011In the following, we propose a simple method to fabricate deformed graphene for easy modification of the graphene–substrate interaction with local control. We choose nanobubbles as the structural ‘knob', that is, an on-off switch for phonon control, and exploit the fact that noble-gas ions can penetrate below the first atomic layer of a graphite surface to form interstitial defects by low-energy impinging1314+ ions, protrusions emerged as a result of certain modifications in graphene but not in Pt(111), as evident in +-ion irradiated graphene can in general be attributed to several origins such as adatom adsorption, vacancy-induced deformation1314+-ion sputtering with subsequent annealing. This is evidenced by the fact that the generally observed resonance peak near the Dirac point, which is characteristic of vacancies in graphene, is indeed absent in the differential conductance (dI/dV) spectrumIn our study, graphene nanoislands were first grown on a Pt(111) substrate. The large-scale topography  as well I/dV spectra of the nanobubbles obtained near their apexes share the common gap-like feature centred at the Fermi level . As shown in K projection occurs at 68–70 meV for all finite-size dots, whose energy range corresponds well with the phonon energy of 67.3 meV in an infinite plane with momentum K. However, the larger the dot size is, the narrower the calculated distribution of the K projection becomes, and this behaviour explains why the depth and the edge slope of the gap-like feature in the dI/dV are observed to increase for larger bubbles. In addition, the calculation of the root mean square amplitude of the phonon modes  maps. The spatial dI/dV map in 2I/dV2 map in I/dV maps because of the gap, and a strong contrast is observed at around ±70 meV in the d2I/dV2 maps, which is manifested as a peak and dip in d2I/dV2 spectra in I/dV and d2I/dV2 maps coincides with that of the nanobubble in the topograph. We also plotted the bias voltages of the largest peak in the d2I/dV2 spectra, Vph, shown in Vph shows little variation from 64 to 70 meV. However, the intensity of phonon signal increases as a function of the distance between the graphene and Pt layers, as plotted in To examine the spatial distribution of excited phonons around graphene nanobubbles, we obtained the spatial dI–V behaviourWe successfully demonstrated phonon excitations in graphene via local control by generating nanobubbles of various sizes by means of Ar-ion irradiation. The reduced graphene–metal interactions due to Ar atoms embedded in between the graphene and metal substrate were shown to promote phonon excitations and enhance electron tunnelling above the phonon energy. We expect that our demonstrated control over phonon excitations down to the nanoscale will pave the way for advancements in graphene-based nano-electromechanical devices such as electrical switches based on the induced nonlinear + ions , and the sample was annealed at 1,000 K to remove additional impurities adsorbed onto graphene. Our STM measurements were performed using a Unisoku low-temperature STM at 2.8 K, and scanning tunnelling spectroscopy was performed using a conventional lock-in technique with a modulation bias voltage at a frequency of 1 kHz and an amplitude of 10 mV.A Pt(111) sample was cleaned by means of repeated cycles of Ar-ion sputtering and annealing under ultrahigh vacuum. Graphene nanoislands were prepared by exposure of the clean Pt(111) surface to ethylene followed by annealing at 1,100 Kab initio simulation package2425−1.Our first-principles calculations are based on the DFT employing the generalized gradient approximation with the projector-augmented-wave method as implemented in the Vienna We computed the Hessian matrix of the atoms within the graphene nanobubble using the adaptive intermolecular reactive empirical bond order (AIREBO) potential for carbonHow to cite this article: Kim, H.W. et al. Nanoscale control of phonon excitations in graphene. Nat. Commun. 6:7528 doi: 10.1038/ncomms8528 (2015).Supplementary Figures 1-7, Supplementary Note 1 and Supplementary References


Charge noise is critical in the performance of gate-controlled quantum dots (QDs). Such information is not yet available for QDs made out of the new material graphene, where both substrate and edge states are known to have important effects. Here we show the 1/f noise for a microscopic graphene QD is substantially larger than that for a macroscopic graphene field-effect transistor (FET), increasing linearly with temperature. To understand its origin, we suspended the graphene QD above the substrate. In contrast to large area graphene FETs, we find that a suspended graphene QD has an almost-identical noise level as an unsuspended one. Tracking noise levels around the Coulomb blockade peak as a function of gate voltage yields potential fluctuations of order 1 μeV, almost one order larger than in GaAs/GaAlAs QDs. Edge states and surface impurities rather than substrate-induced disorders, appear to dominate the 1/f noise, thus affecting the coherency of graphene nano-devices. There has been no report on the charge noise for graphene nano-devices where the carrier channel size is of 10-nm order although researchers have fabricated various graphene nanostructures including single graphene quantum dots (GQDs)192021222324262728292 was etched away to a depth of about 50 nm. We estimate our graphene nanoribbon was suspended about 50 nm above the substrate. By tilting the sample holder of the SEM to a specified angle, we can observe the nano-device from the side to check whether the graphene nanoribbon was suspended. As shown in After dipping the sample in the BOE for 40 seconds, SiOWe used the standard lock-in method to probe the electronic signals to make the suspended nanoribbon work in the Coulomb blockade region. We applied a dc voltage to the back gate to tune the Fermi energy of the graphene. The V-shaped current-voltage relation was observed, and a transport gap was found in the range 0.5–6.0 V . We thenWe measured the current fluctuation using a spectrum analyzer (SR785). A schematic of the circuit used is shown in I(f) is the noise spectrum measured. SCB(f) is the background noise induced by the measurement system. Here we used the spectrum at C as SCB.We calculated the magnitude of the current fluctuation ΔI by integrating the spectrum from 1 to 9 Hz; SD = 200 μV to the source and measured the current from the drain. Changing the back gate voltage from 4.283 to 4.297 V, we obtained a Coulomb peak. I at different back-gate voltages along the Coulomb peak and calculated the integral in −1|dI/dV|Δε given in Ref. Next, we applied a dc voltage VThe potential fluctuation Δε, calculated from the peak in We also measured the current noise of regular (unsuspended) graphene nanoribbon devices for comparison. The regular graphene device was fabricated in a similar way as described above. The only difference was that the regular samples were not dipped in BOE after making electrodes. Similar noise measurements of regular devices were performed under the same experiment conditions. To investigate the influence of the temperature on the potential fluctuation, we measured the same Coulomb peak at different temperatures, from 240 mK to 1 K. From 2* in graphene nano-devices. Notably, we also used the method described in Ref. We measured different Coulomb peaks from several devices, both suspended and unsuspended. For E, the density of disorders in the substrate is nS. The effect of edge states and substrate on the low frequency noise of the device is proportional to S. In the GFET experiment, the removal of the substrate decreases the noise by one order of magnitudeIt is well known that the edge states can greatly affect the properties of graphene devices. The edge states change the electron distribution, resulting in the formation of puddles in graphene nano-devices3738Although this simple model can qualitatively explain the present experiment, more studies are still needed to investigate how the edge states or surface impurities affect the 1/f noise, and how to decrease the noise level in the graphene devices.In summary, we have fabricated both suspended and unsuspended graphene nano-devices and measured their 1/f noise along the Coulomb peaks to obtain the charge noise level. Suspending the QD from the substrate had no observable effect on the charge noise of a GQD. The edge states closely surrounding these nano-devices are argued to increase the charge noise to 1 μeV and become the dominant charge noise source instead of that from the substrate. More studies are needed to improve the electronic performance and increase the quantum coherence of future graphene nano-devices.2 layer away. To avoid graphene from being wrinkled, we used a critical-point dryer to dry the sample instead of blowing with N2.Graphene flakes were produced by mechanical cleaving of bulk graphite and deposited on a highly doped silicon substrate covered by 100 nm of silicon dioxide. The doped silicon substrate worked as a back gate. Graphene flakes were found using an optical microscope, and few-layer flakes were selected using Raman spectroscopy40X.X.S. fabricated the samples. X.X.S., H.O.L., J.Y., T.Y.H., G.C. and G.P.G. performed the measurements. X.X.S., M.X., T.T. and G.C.G. provided theoretical support. Data are analyzed by X.X.S., H.O.L. and J.Y. The manuscript is prepared by X.X.S., H.W.J. and G.P.G., G.P.G. supervised the project. All authors contributed in discussing the results and commented on the manuscript.Supporting information


The ability to control light-matter interaction is central to several potential applications in lasing, sensing, and communication. Graphene plasmons provide a way of strongly enhancing the interaction and realizing ultrathin optoelectronic devices. Here, we find that photoluminescence (PL) intensities of the graphene/GeSi quantum dots hybrid structures are saturated and quenched under positive and negative voltages at the excitation of 325 nm, respectively. A mechanism called plasmon-gating effect is proposed to reveal the PL dependence of the hybrid structures on the external electric field. On the contrary, the PL intensities at the excitation of 405 and 795 nm of the hybrid structures are quenched due to the charge transfer by tuning the Fermi level of graphene or the blocking of the excitons recombination by excitons separation effect. The results also provide an evidence for the charge transfer mechanism. The plasmon gating effect on the PL provides a new way to control the optical properties of graphene/QD hybrid structures. It has been found that by electric gating and thermal annealing, the dynamic conductivity of graphene device would change largely, and the Fermi energy of graphene is effectively tuned. As a result, the guided light can be modulated within a broad operation spectrum212278Thus, we naturally consider whether the SPP enhanced PL properties of the graphene/GeSi QDs hybrid structure can be controlled by introducing an external electric field. It is also easily considered whether the PL quenching, based on the carrier transfer or the energy transfer of excitons, can be directly indicated by tuning the graphene Fermi level with a bias voltage. The controlling PL properties of QDs are very essential to its application in improving optical properties. In this work, we fabricate the graphene/GeSi QDs hybrid structure devices to explore their optical properties by adding external electric field. It is indicated that the vertical electric field nonlinearly determines the enhancement or quenching of PL intensity at the different excitation wavelengths. At the excitation of 325 nm laser line, which is the resonant excitation wavelength of graphene plasmon−1) to G-band (~1580 cm−1) is larger than two. The 2D-band is quite shape and cannot be decomposed into two or more components. The characteristics indicate that the graphene on the GeSi QDs is monolayer one. And the low intensity of D-band (1341 cm−1) demonstrates the good quality of graphene sheet28The schematic diagram of the graphene/ten-layer GeSi QDs hybrid structure device demonstrates that the positive voltage was added on the Al back electrode, and the In point electrode was connected with graphene on the top of the device . Figure In our previous publicationNext, the PL spectra of graphene/GeSi QDs hybrid structures are obtained with the other exciting wavelengths for different bias voltages. At the exciting wavelengths of 405 and 795 nm, it is well known that there is no SPP enhanced absorption in graphene2 layer between the first layer GeSi QDs and the graphene. Thus, the SPP can influence the topmost layer GeSi QDs. The resonant excitation of graphene SPP can enhance the absorption at surface region so that the generation of electron-hole pairs in the topmost layer QDs is enhanced. For the hybrid structure, there are two competing mechanisms. One is the SPP absorption mechanism to enhance the PL intensity, and another is the electron transferring from QDs to the graphene, which reduce the PL intensity. Under the positive voltage, the oblique band alignment shown in At the excitation of 325 nm and under positive biased voltage, the results look quite interesting. From Similar to photogating effect in the graphene covered PbS QDs structure, in which the photogenerated charges can transfer to the graphene and the oppositely charged carriers remain trapped in the QD layer under the build-in field at the QD/graphene interfaceIn summary, we have fabricated a graphene/GeSi QDs hybrid structure device and measured the PL properties under varying bias voltages at different excitation wavelengths. At all three exciting wavelengths, the PL intensity of the hybrid structure is found to be quenched with increasing negative biased voltage on the graphene/QDs structure, which can be explained by the electron transfer model. Under positive biased voltage, different dependences of PL intensity of the hybrid structure on the voltages are observed at three excitation wavelengths. At the excitation of 325 nm laser line, a plasmon-gating mechanism is proposed for the observed PL intensity dependence on the positive biased voltage. At the excitations of 405 and 795 nm, the PL intensity of the hybrid structure is found to be quenched with increasing positive bias voltage, which is explained by excitons separation effect. The gating of the plasmon resonance excitation and its effect on the photoluminescence provides a new way to control the optical properties of graphene/QD hybrid structures.10 layers ordered GeSi QDs sample was grown on pit-patterned Si (001) substrates with periods of 100 nm by solid source Molecular Beam Epitaxy. Large-scale graphene layer was fabricated by chemical vapor deposition (CVD) on a Cu substrate. The structure of graphene was confirmed by Fourier transform infrared (FTIR)-Raman spectra with the excitation of 325 nm laser line. Surface morphology of the sample was characterized using atomic force microscopy (AFM). Then the monolayer graphene was transferred on to the surface of GeSi QD sample to form graphene/GeSi QDs hybrid structures with the aid of polymethylmethacrylate (PMMA).2. Two electrical lines from the probes were pressed on the In electrode and the back side. The vertical electric fields can added to the sample in the vacuum chamber using an external 2400 Keithley Source Meter. We provide stable applied bias voltage from −100 V to 100 V.In order to add the vertical electric field on the hybrid structure, the In and Al contacts are deposited as the front and back electrodes. The back electrode was formed by thermal evaporation of 300 nm Al on the back of the silicon wafer, and the front electrode was formed by alloying In contact at the top of graphene layer with about 4 mm2. The PL spectra were recorded by an extended InGaAs detector using the standard lock-in technique.The PL measurements under different voltages were performed in a He-flow cryostat with the 325 nm line of HeCd laser, and 405 and 795 nm lines of diode lasers as the excitation source. The spot size of laser can be focused to 1 mmHow to cite this article: Chen, Y. et al. Plasmon-gating photoluminescence in graphene/GeSi quantum dots hybrid structures. Sci. Rep.5, 17688; doi: 10.1038/srep17688 (2015).


Graphene is a flat monolayer of carbon atoms, arranged in a two-dimensional hexagonal structure, with extraordinary electrical, thermal, and physical properties. Moreover, the molecular structure of graphene can be chemically modified with molecules of interest to promote the development of high-performance devices. Although carbon derivatives have been extensively employed in industry and electronics, their use in regenerative medicine is still in an early phase. Study prove that graphene is highly biocompatible, has low toxicity and a large dosage loading capacity. This review describes the ability of graphene and its related materials to induce stem cells differentiation into osteogenic, neuronal, and adipogenic lineages. An outburst of research on regenerative medicine has recently emerged to develop nanostructured materials as smart interfaces to be used for cellular studies and regenerative medicine. Tissue regeneration is a demanding field in terms of development of biomaterials since it requires a variety of fabrication scales, ranging from signal transduction levels to macroscopic tissue recapitulation. Current nanoscale research is focusing primarily on new materials that might be manufactured at high production volume and thus be associated with significant human application. The final goal is a better understanding of the complexity that entails the native extracellular matrix (ECM) through the creation of in vitro models, leading to engineered scaffolds specifically designed to modulate cells differentiation. Many authors have demonstrated that cell shape, morphology, attachment, proliferation, and migration can be controlled by cell-material interactions –4. The rIn the search for brand new factors which may have an influence on cell behavior, much attention has been recently given to environmental components ,7, and pAlthough carbon derivatives have been extensively employed in industry and electronics, their use in regenerative medicine is still in an early phase . The strThe exceptional properties of graphene and its potential different applications have led to the development of composite materials that include few-layer-graphene (FLG), ultrathin graphite, graphene oxide (GO), reduced graphene oxide (rGO), and graphene nanosheets (GNS), comprising a broad set of “graphene-family nanomaterials” (GFNs) Figure  15–17].–17.15–17Some of them, like GO substrates, have been already demonstrated to stimulate human MSCs to differentiate into adypocites ,18, to iBone tissue engineering promises to restore bone defects that are caused by severe trauma, congenital malformations, etc. Many researchers are studying the ways to confer a pro-osteodifferentiation or osteoinductive capability on implants or scaffold materials, where osteogenesis of seed cells is promoted. Graphene provides a new kind of coating material that may confer the pro-osteodifferentiation capability on implants and scaffold materials by surface modification. Here, we review recent studies on the effects of graphene on surface modifications of implants or scaffold materials. The ability of graphene to improve the biological properties of scaffold materials, and its ability to promote the adhesion, proliferation, and osteogenic differentiation of MSCs or osteoblasts have been demonstrated in several studies .2) and graphene-coated SiO2 [2 surface. The results were supported by fluorescent imaging, revealing that in the graphene substrate the initial number of osteoblasts almost duplicated, compared to a 1,5 increase factor in the SiO2 film. Nonetheless, these initial observations tended to be controversial, considering the study of Nayak et al. [2, it did not demonstrate to influence the shape or structure of seeded cells compared to uncoated surfaces. Furthermore, these authors investigated the osteogenic potential of graphene on MSCs. MSCs cultured on uncoated Si/SiO2, PDMS and PET surfaces showed a CD-44 positive staining and a complete negative osteocalcin (OCN) staining. On the other hand, OCN was significantly represented when cells were cultured on the same graphene-coated surfaces  in loading and releasing bone morphogenic protein type 2 (BMP-2) . The ostThe osteogenic differentiation potential of MSCs on graphene substrates was confirmed also by the study of Crowder et al., where a 3D graphene structure was used to induce osteogenic differentiation . The autin vitro co-culture of hBMMSCs and in vivo implantation. The nanofibrous structure itself demonstrated significant enhancement in cell adhesion, proliferation and osteogenic differentiation of hBMMSCs, and with the incorporation of CNMs, the composite nanofibrous scaffolds further promoted osteogenic differentiation of hBMMSCs significantly. Between the two CNMs, graphene showed stronger effect in promoting osteogenic differentiation of hBMMSCs than CNT. The results of in vivo experiments revealed that the composite nanofibrous scaffolds had both good biocompatibility and strong ability in inducing osteogenesis. CNMs could remarkably enhance the expression of osteogenesis-related proteins as well as the formation of type I collagen. Similarly, the graphene-containing composite nanofibrous scaffolds demonstrated the strongest effect on inducing osteogenesis in vivo. These findings demonstrated that CNMs-containing composite nanofibrous scaffolds were obviously more efficient in promoting osteogenesis than pure polymeric scaffolds [The effect of graphene on MSC osteogenic commitment has been also studied by Duan et al.  who compcaffolds .The same positive results have been observed by Tavarty et al.  that hypIn vitro cytocompatibility of the GC1,5 coating was evaluated using a hBMMSCs culture system. The proliferation and ALP, osteopontin and OCN osteogenesis-related gene expression of the cells on the GC1,5 coating did not deteriorate with the adoption of graphene. Conversely, even better adhesion of the hBMMSCs was observed on the GC1,5 coating than on the pure CS coating. All of the results indicate that the GC1,5 coating is a good candidate for load-bearing implants [Owing to the superior mechanical properties and low coefficient of thermal expansion, graphene has been widely used in the reinforcement of ceramics. Xie et al.  studied implants .Inducing human NSCs to differentiate into neurons is a critical challenge to reach an important biomedical goal, since a promising opportunity in therapies for neural regeneration could arise ,27.Park et al. tested the graphene substrate as a promoter of human NSCs differentiation into neurons . The mosOther results indicating that graphene-based substrates can promote neural differentiation came from the study by Hong et al. . After cIn the attempt of influencing in a significant way stem cell differentiation, several declination of graphene scaffolds were studied. Wang et al. tested MSCs on fluorinated sheets of graphene, observing a strong enhancement of neuronal differentiation when compared to cells seeded on graphene . This inThe study by Yang et al. investigated the capability of GO, graphene and CNTs to induces dopamine neural differentiation of mouse embryonic stem cells (ESCs) . ESCs weAnother important characteristic of graphene could be represented by its capability to form a functional neural network. In the study by Tang et al., neurospheres were seeded on graphene substrates and after a 14 days culture the process of network formation was made clear by beta-tubulin immunostaining . Newly f2, GO/TiO2, and rGO/TiO2), with and without flash photo stimulation [2 substrate, by a rate of 48% in the GO/TiO2 substrate and by a rate of 24% on the TiO2 substrate. No differences were detected without flash stimulation. When the differentiation rate was analyzed it was found that the rGO/TiO2 substrate was the more beneficiary of the flash photo stimulation: an 88% decrease in the ratio of glial cells and an 81% increase in the ratio of neuronal cells. In the other 2 substrates the difference was less evident: 25% of neuronal cells increase rate on the GO/TiO2 substrate and 15% on the TiO2 substrate. Overall, flash photo stimulation of graphene-based substrate provide both a greater cell proliferation and a greater human NSCs differentiation into neurons rather that glia cells. However, these results could be obtained when the flash photo stimulation of human NSCs was at an optimum concentration of a biocompatible hole scavenger, and at an optimum flash intensity.Stimulating human NSCs to differentiate into neurons rather than glia is a another key point in order to obtain a neural regeneration result . The stumulation . When thFor central nervous system (CNS) regeneration, the selective differentiation of NSCs into either neurons or oligodendrocytes (as opposed to astrocytes) is highly desirable. Several approaches have been employed to guide differentiation into neurons, however, oligodendrocyte differentiation has proven to be much more elusive, resulting in only a small percentage of the differentiated cell population. The primary approach to guide oligodendrocyte differentiation has focused on either developing culture media containing a combination of growth factors or the forced expression of key oligodendrocyte-promoting transcription factors. In this view the developing a biomaterials-approach to achieve efficient differentiation of NSCs into mature oligodendrocytes, while eliminating the potential adverse or variable side-effects from growth factors and viral gene vectors, would be highly beneficial.Shah et al.  developeGO based nanoparticles has been used also to drive the commitment of ESCs into dopamine neurons. Yang et al.  studied in vitro and in vivo, then lead to other biological effects, as expected of any foreign particulate. Current studies mostly focus on the pulmonary inflammation caused by nanomaterials insults, while little is known about the neuroinflammatory effects. Given that some nanomaterials  have been attempted to be used in neuroscience, the neuroinflammation should be considered. External insults range from hypoxia and ischemia to a number of bacterial and viralinfections, all of which elicit a characteristic neuroinflammatory reaction in the brain. Microglia, astrocytes, and peripheral macrophages are key players mediating this response. In the brain, most of the damage caused by nanomaterials is mediated by the microglia, a macrophage-like, phagocytic cell that is normally inactive unless confronted by potentially damaging xenobiotics. In response to certain cues such as brain injury or immunological stimuli, however, microglia are readily activated. Graphene has been at the forefront of nanotechnology and advanced materials sciences due to its intriguing physical and chemical features. Especially, it has been utilized in a variety of biomedical applications. Recently, Li et al. [in vitro culturing and justify whether this graphene-induced neuroinflammatory effects are detrimental or beneficial to the neural cells. The importance of this work is the elucidation of the pro- and/or anti-inflammatory effects of graphene and pave the way for the applications of graphene in biomedicine. The graphene, especially 3D graphene, supported microglia growth and showed comparable biocompatibility to the commercial tissue culture polystyrene substrates. Despite of the similar proinflammatory responses in the microglia without lipopolysaccharide (LPS) activation, 3D graphene evoked much milder neuroinflammation in the microglia after LPS activation in comparison to 2D graphene, suggesting that the topographical structures of the materials might affect the inflammatory behaviors. Furthermore, the unique topographical structures of 3D-graphene may restrict the morphological transformation of microglia under over-activation, leading to the anti-inflammatory effects.Novel important applications of graphene in neuroscience has been found in the end by Song at al. , who stui et al.  discoverGraphene and GO substrates have been also used for investigating their effects on the adipogenic differentiation of MSCs. In particular, in the study of Lee et al., MSCs were plated on graphene or GO sheets in presence of adipogenic differentiation medium for 14 days . The celKim et al. evaluated GO potential in differentiating human adipose-derived stem cells (ADSCs) into adipocytes, demonstrating a higher adipogenesis on the GO substrate when compared to the control (tissue culture polystyrene) . This stIn regenerative dentistry, stem cell-based therapy often requires a scaffold to deliver cells or growth factors to the injured site. GO and silk fibroin (SF) are promising biomaterials for tissue engineering as they are both non toxic and promote cell proliferation. A field that could be improved by the availability of an effective film scaffold would be the reparation of periodontal tissues. The periodontium, which is composed of four dental tissues , is constantly maintained by periodontal ligament stem cells (PDLSCs) owing to their great capacity of differentiation into cementoblasts, odontoblasts, and fibroblasts. PDL plays a key role in the attachment of teeth to the jaw; in the most drastic cases of periodontitis, which is associated with a chronic inflammation process, PDL destruction could lead to the loss of the tooth . With thAs well reported above, graphene has drawn attention as a substrate for stem cell culture and has been reported to stimulate the differentiation of multipotent adult stem cells. Recently, Lee et al.  reportedThe same results have been reched by Park et al.  that demThe successful reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) by Takahashi and Yamanaka in 2006  was seenIn an interesting study by Yoo et al. , the autThe generation of ESCs-like cells from somatic cells by ectopic expression of defined factors is an approach commonly known as cell reprogramming. Moreover, because iPSCs generation is known to be a multiple-step process mediated by overexpression of the transcription factors Oct4, Sox2, Klf4, and cMyc, the generation of iPSCs is very inefficient, and cell reprogramming is considered a stochastic process in which successive barriers must be overcome to reach a state of pluripotency. In particular, one of the first noticeable changes during the reprogramming of somatic fibroblasts is their transformation into tightly packed clusters of rounded cells in a process that resembles a mesenchymal-to-epithelial-transition (MET). More interestingly, recent studies found that microtopography substrate affects the MET, improving reprogramming efficiency. The authors examinated whether somatic fibroblasts should be efficiently reprogrammed into the pluripotent state on graphene-based substrate. Fitstly they carachterised the monolayer graphene film by Raman spectroscopy, then they studied whether the graphene substrate enhances cell reprogramming by seeding Oct4-GFP knock-in (KI) mouse embryonic fibroblasts (MEFs) onto both the control and graphene-coated substrate in MEF medium. One day after plating, MEFs were transduced using specific vectors expressing Oct4, Sox2, Klf4, and cMyc transcription factor. The MEFs plated on the graphene substrate began to form colonies 10 days after viral infection, exibiting a significant increase in colonies undergoing reprogramming on graphene substrate. To quantify the reprogramming efficiency, they performed FACS analysis for Oct4-GFP-positive iPSCs derived from Oct4-GFP KI MEFs in both the control substrate and graphene-coated substrate. Fifteen days after doxycycline (dox) induction, the graphene substrate cultures had a significant increase in the number of GFP-cells and quantitative real-time PCR analysis showed that pluripotency marker genes, including Oct4, Nanog, Sox2 and Esrrb were markedly elevated in graphene-coated substrate cultures compared to uncoated substrate cultures. The pluripotent state of graphene-induced iPSCs was assessed by immunostaining of pluripotency marker. Consistent with these results, they observed that the graphene-coated substrate significantly increased the number of Oct4, SSEA1 and Nanog colonies. Moreover, in order to analyze the differentiation potential of graphene mediated iPSCs, their capacity for direct differentiation into the cell types of the three germ layers has been tested. Immunocytochemical staining and real-time PCR showed that the differentiated cells were positive. In the end the further developmental potency of the graphene induced iPSC lines was investigated by SCID mice. Four weeks after injection, teratomas were readily visible and histological analysis confirmed the presence of cell types derived from all three embryonic germ layers validating the pluripotency of these graphene induced iPS cells. In order to examine whether graphene substrate affects chromatin state during reprogramming, they measured the enrichment level of histone modifications that mark active  in the graphene induced cell reprogramming. Surprisingly, they found a dramatic increase of H3K4me3 expression on graphene substrate and that the transcription start sites of Oct4 and Nanog were enriched for H3K4me3 in graphene surface-induced reprogramming cultures. Taken together, these results suggest that the graphene-coated substrate specifically promotes the MET process in cell reprogramming without affecting the EMT process. In this study, the autors showed that the epigenetic reprogramming of somatic fibroblasts is enhanced on graphene substrate providing a potential method for the efficient generation of iPSCs. Interestingly, they found a specific induction of MET by the graphene substrate during reprogramming and an accumulation of H3K4me3, which facilitates increased Oct4 and Nanog occupancy. These data indicate a unique role for graphene substrates in facilitating cell reprogramming .Carbon nanoparticles such as zero dimensional (0D) fullerenes, one dimensional (1D) CNTs, and recently two dimensional (2D) graphene have been investigated for applications in therapeutics, bioimaging, and regenerative medicine. MSCs are currently being widely investigated to repair, regenerate, and restore damaged tissues. Nanoparticles have been employed to deliver growth factors or genes into MSCs to manipulate their differentiation . The devThe literature on the biological interactions of graphene family is growing rapidly, and includes studies primarily motivated by biomedical applications, and environmental health and safety. As with other biomaterials, the issue of potential toxicity arises not only in biomedical applications, but also in non-biomedical products where unintended occupational, consumer, and environmental exposures can occur. The exact mechanism of GO and the derived composites in SCs differentiation is still unresolved. It has been generally hypothesized that the surface characteristics of graphene family nanomaterials such as nanotopography, surface stiffness, and large absorption capacity influence the molecular pathways that control the fate of stem cells. Both G and GO were reported acting as preconcentrators for chemicals, proteins and growth factors on their surface to promote cell differentiation.In this view, we have reported a review of the international literature to produce a state-of-the-art update of graphene applied to tissue engineering and stem cells.Many studies confirmed that graphene and its related materials are able to induce human stem cells differentiation into specific lineages. Materials coated with graphene or GO or even 3D graphene foam were capable to guarantee viability and to induce osteogenic differentiation of stem cells when compared with traditional substrates or scaffolds ,14,21,22Even if we are still uncertain about potential in vivo applications, mostly due to open questions around toxicity, graphene clearly appears to be a step forward in the field of tissue engineering.In this light it is interesting the results found by Chen et al. that express an expert opinion based on scientometric patterns revealed by CiteSpace without prior working experience in the regenerative medicine field. Emerging trends and new developments have been identified based on structural and temporal properties derived from the relevant publications. The detected surge of the keyword graphene in the literature of regenerative medicine led the authors to investigate the nature and context of its use in regenerative medicine. The investigation revealed a rapidly increasing number of studies that specifically used graphene and GO in regenerative medicine research because of their desirable surface properties for, among other applications, culturing and maintaining SCs. Similarly a detected burst of citations provides insightful guidance for navigating through the fast-changing .ad hoc sample names.There is also an emerging literature on potential health risks. Despite the popular image of graphene as a large-area substrate coating, many graphene-family materials are dry powders at some point in their processing, and in this form pose the most significant exposure risk through inhalation. Of particular concern are FLG samples directly following the thermal exfoliation step or after washing and drying. There is need for measurement of airborne dust levels in research laboratories, and in pilot and full-scale manufacturing facilities. GFNs are high-surface-area materials with corresponding high potential to cause adsorptive and quenching artifacts in biological assays . Adsorpt


In this study, a CMOS compatible capacitive humidity sensor structure was designed and fabricated on a 200 mm CMOS BEOL Line. A top Al interconnect layer was used as an electrode with a comb/serpent structure, and graphene oxide (GO) was used as sensing material. XRD analysis was done which shows that GO sensing material has a strong and sharp (002) peak at about 10.278°, whereas graphite has (002) peak at about 26°. Device level CV and IV curves were measured in mini-environments at different relative humidity (RH) level, and saturated salt solutions were used to build these mini-environments. To evaluate the potential value of GO material in humidity sensor applications, a prototype humidity sensor was designed and fabricated by integrating the sensor with a dedicated readout ASIC and display/calibration module. Measurements in different mini-environments show that the GO-based humidity sensor has higher sensitivity, faster recovery time and good linearity performance. Compared with a standard humidity sensor, the measured RH data of our prototype humidity sensor can match well that of the standard product. With the consumer market growth, requirements for high performance and low cost MEMS/sensors are increasing, especially for digital multimedia products [etc. Recently, humidity sensors have been successfully integrated into high-end mobile phone products including the Samsung Galaxy S4, and their market will increase dramatically with the growing consumer electronics demands.With the developing of the Internet of Things and sensor networks, MEMS/sensor “consumerization” has begun to emerge, and more and more new applications of MEMS/sensors have begun to be found in consumer products such as mobile phones, PAD, products ,2. HumidBased on their measurement units, humidity sensors can be divided into two types: relative and absolute humidity sensors. According to the operating principles, these are three main types: Capacitive, resistive, and thermal conductivity humidity sensors. Most humidity sensors are capacitive RH sensors because of their low cost, linearity, minimal long-term drift and hysteresis .2 and sp3 hybridized carbon atoms which can be considered as insulators [etc.Graphene is one of the most promising materials developed in this century with its exceptional mechanical, thermal and electrical properties ,5,6,7. Gsulators . GO is ssulators ,10,11,12sulators ,15,16,17In this work, a capacitive humidity sensor was designed and fabricated with GO as sensing material. The capacitance of the sensor device was increased about four times from ~22.5% RH to ~85% RH, which shows excellent sensitivity. After integrating the sensor device with a dedicated readout ASIC, a prototype humidity sensor was built and its measured data was compared with that of a standard humidity sensor.The sensor structure was designed and fabricated with a comb/serpent capacitor structure by a standard 0.35 μm CMOS compatible process, as shown in After wafer dicing was finished, and the sensor was fabricated by filling GO material in the structure. In this post CMOS process, a GO-based water dispersion solution was used as filling material, and after the filling process the sensor was thermally treated to remove the water. XRD was used to check the sensing material properties, and CV/IV data was measured in different environments to check the leakage and capacitance properties.A prototype humidity sensor was designed and built by integrating the sensor with a dedicated readout ASIC and display/calibration module at the PCB level. A standard frequency-voltage convertor  was used as readout circuit to extract the capacitance variation by the RC oscillation mechanism. The prototype sensor was then evaluated in different environments, and all the measured data was compared with a standard humidity sensor  as shown in All the measurements were done at a room temperature of 25 °C. CV was measured by a 4284A instrument  and IV was measured by an Agilent 4156C device. This study adopted a very convenient way to measure and calibrate humidity sensors by using saturated salt solutions. As shown in The RH of the atmosphere above the saturated salt solution in the sealed chamber was fixed at a given temperature, and it can cover almost the entire range of relative humidity, as shown in A standard 0.35 μm CMOS BEOL process was used to fabricate the comb/serpent capacitor structure. A BEOL Al metal layer was used as capacitor electrode with line/space/height = 0.78 μm/0.54 μm/0.8 μm. The structure was designed with consideration of least fringing field and parasitic capacitance, and PAD was formed by the same metal layer. A top-view photo of the structure can be seen in −11 f, and a very good uniformity can be obtained with a standard deviation of ~1.76E−13 f and NU% of ~ 1.41%. From the IV data, the structure shows a very good leakage control with a max leakage current of ~5E−14 A.After wafer fabout, a CV and IV mapping test was done to check the uniformity and leakage performance of the structure. As can be seen in After wafer dicing, the sensor was fabricated by filling GO material in the sensor structure. A GO-based water dispersion solution was used as filling material, and after the filling process the sensor was treated in a baking oven at a temperature of 60 °C for about 50 min to remove the water. Cross-sectional SEM was done to check the filling performance, and the GO filling performance can meet the sensor requirements as can be seen in 3 structure network. Because of the existence of oxygen functional groups and some other structural defects, the interlayer distance of GO is greatly increased [GO film was prepared on a blanket substrate to do XRD analysis. Compared with graphite, GO has a variety of reactive oxygen groups, such as carboxylic acids (COOH), carbonyls (C=O), epoxides (–O–) and hydroxyls (–OH), which are linked to a C-spncreased ,13,14,15−11 A with a comb/serpent electrode length of ~165 mm at about 50% RH level, which can meet well the sensor application requirements.Leakage is very critical to any sensor application with a capacitive sensing mechanism, because more leakage current will make capacitance measurement inaccurate and cause the device to malfunction. Leakage of the sensor device was measured in voltage sweeping mode from 0 V to 5 V. As shown in Capacitance was measured at frequency of 100 kHz by an Agilent 4284A instrument. C/C0 ratio is plotted in To evaluate the potential value of GO material in humidity sensor applications, a prototype humidity sensor was designed and built by integrating the sensor with a dedicated readout ASIC and a display/calibration module on the PCB level. A standard Analog Microelectronics CAV444 frequency-voltage convertor was used as a readout circuit to extract the capacitance variation caused by the RC oscillation mechanism. A schematic circuit block of the prototype sensor and block diagram of the frequency-voltage convertor are shown in To build the prototype sensor, the sensor device was firstly wire-bonded with the CAV444 on the PCB level, which can convert the change of capacitance to voltage. As can be seen in After that, the prototype humidity sensor was built by further integrating a LCD display and calibration module, which can be seen in After linear calibration, the prototype sensor was used to measure the RH value in environments at different RH levels. The measured data was then compared with that of a standard humidity sensor, as shown in Response/recovery time was defined as the time (τ) required to achieve ~63% value of a humidity step function. Recovery time was measured by a simple method as shown in In this work, a CMOS compatible capacitive humidity sensor structure was designed and fabricated on a 200 mm CMOS BEOL Line. BEOL Al with a comb/serpent structure was used as electrode, and GO was used as sensing material. The sensor device shows excellent sensitivity and faster recovery time than a standard sensor. A prototype humidity sensor was designed and built by integrating the sensor device with a dedicated ASIC at the PCB level, and the output value of the prototype sensor was shown to match well that of a standard humidity sensor.


Streptococcus pneumoniae infection are key in devising strategies towards the development of better vaccines and drugs. To that end, in this study we investigated the role of S. pneumoniae and its surface antigen Pneumococcal surface protein A (PspA) in modulating the expression of co-stimulatory molecule Programmed Death Ligand 1 (PD-L1) expression on dendritic cells (DCs) and the subsequent effects of increased PD-L1 on key defence responses. Our data indicate that stimulation of DCs with PspA increases the surface expression of PD-L1 in a time and dose dependent manner. Characterization of mechanisms involved in PspA induced expression of PD-L1 indicate the involvement of Toll-Like Receptor 2 (TLR2) and calcium homeostasis. While calcium release from intracellular stores positively regulated PD-L1 expression, calcium influx from external milieu negatively regulated PD-L1 expression. Increase in PD-L1 expression, when costimulated with PspA and through TLR2 was higher than when stimulated with PspA or through TLR2. Further, knockdown of TLR2 and the intermediates in the TLR signaling machinery pointed towards the involvement of a MyD88 dependent pathway in PspA induced PD-L1 expression. Incubation of DCs with S. pneumoniae resulted in the up-regulation of PD-L1 expression, while infection with a strain lacking surface PspA failed to do so. Our data also suggests the role of PspA in ROS generation. These results suggest a novel and specific role for PspA in modulating immune responses against S. pneumoniae by regulating PD-L1 expression.Pneumonia leads to high mortality in children under the age of five years worldwide, resulting in close to 20 percent of all deaths in this age group. Therefore, investigations into host-pathogen interactions during  S. pneumoniae is the number one cause of bacterial pneumonia and can also cause meningitis, sepsis and otitis media. In some developing countries Streptococcus pneumoniae can account for over 50% of all pneumonia deaths [S. pneumoniae based on the structure of the polysaccharide capsules [S. pneumoniae also possess various surface associated proteins that contribute to its virulence; many of which are able to elicit measurable protection in mice e. g. Pneumococcal surface protein A (PspA) [Pneumonia and pneumonia related illnesses are a major cause of mortality in children under the age of five years worldwide, with close to 20 percent of all deaths in this age group . S. pneua deaths . There acapsules . A 7-valcapsules . The capA (PspA) , 8, 9.PspA, a serologically variable, cross-reactive, cross-protective protein is present on almost all strains of pneumococci and has been shown to be a promising candidate antigen for a protein-based vaccine –12. Anti+ and CD8+ T cells [Staphylococcus aureus triggered conversion of neonatal CD4+ T cells into FOXP3+ CD25+ CD127low T cells [S. aureus modulates T cell responses via TLR2 and PD-L1 [-/- mice respond inadequately to Mycobacterium tuberculosis infection [Burkholderia pseudomallei impairs T cell function [The costimulatory molecule Programmed Death Ligand-1 (PD-L1) is constitutively expressed and its surface expression is upregulated on murine hematopoietic cells  and non-hematopoietic cells  upon stimulation. It has been documented that PD-L1 interacts specifically with B7-1 to inhibit T cell proliferation . The int T cells . The PD- T cells . S. aurend PD-L1 . T cellsnfection . PD-L1 efunction .S. pneumoniae to escape phagocytic killing by DCs and for eliciting optimal proinflammatory cytokine production [S. pneumonniae induces apoptosis in bone marrow derived DCs via two mechanistically different pathways. Pneumolysin dependent but caspase independent and delayed onset but caspase dependent mechanism accompanied with DC maturation [S. pneumoniae during pneumococcal pneumonia [S. pneumoniae by DCs [S. pneumoniae or its antigens in modulating PD-L1 expression on DC is not reported. In this study we investigated the role of S. pneumoniae and its antigen PspA in regulating the surface expression of PD-L1 on DCs. As DCs are known for their critical role in regulation and modulation of the host immune response against bacterial infections, in this study, we characterized the role of S. pneumoniae infection in regulating PD-L1 expression. Our data indicate that PspA induces the expression of PD-L1 on mouse bone marrow derived DCs. Results also point towards a role for the route of calcium entry and Toll-Like Receptor (TLR) 2 in mediating PD-L1 expression.DCs are crucial for innate and adaptive immune response against bacterial pathogens. In recent years, several groups have studied how pneumococcal virulence factors modulate DC function. The precise role of PD-1:PD-L1 pathway in dampening the T cell response is still unclear in pneumococcal infections. The role of PavA during pneumococcal infection has been investigated and it was demonstrated that it is essential for oduction . S. pneuturation . Furtherneumonia . Cao et e by DCs . Clp coue by DCs . HoweverAnimal experiments were done with approval and in strict accordance with the guidelines of the National Institute of Immunology Institutional Animal Ethics Committee (IAEC#327/13). The procedures used in the study were carried out by well-experienced competent workers trained to perform them. All efforts were made to minimize suffering and pain.S. pneumoniae strain D39 (capsular type 2), R6 (ATCC BAAA-255) and JY2008 (ATCC 55143) were used in the study [S. pneumoniae for 3h followed by addition of 40 μg/ml of gentamycin  and DCs were incubated at 37°C for an additional 24h.Virulent he study . JY2008 he study  and mainPhycoerythrin-tagged antibody against mouse PD-L1 were procured from BD Biosciences USA. Recombinant mouse Granulocyte macrophage colony stimulating factor (GM-CSF) was procured from R & D Systems (USA). Antibodies to pAkt, Bcl2, Bax and β-actin, specific and control siRNAs against mouse genes, and Luminol kit for chemiluminescence detection were purchased from Santa Cruz Biotechnology, USA. Chemicals U0126, 3, 4, 5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), ethyleneglycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) and diphenyleneiodonium (DPI) were purchased from Sigma Chemical Co., USA.+ cells were depleted following Magnetic Assisted Cell Sorting. Cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 0.05 M 2-mercaptoethanol, 1 mM sodium pyruvate plus 15 ng/ml GM-CSF for 72h. Recombinant PspA was expressed and purified as described before [3Csk4, TLR4 was stimulated with 0.1 μg/ml lipopolysaccharide (LPS) and TLR7 was stimulated with 1.0 μg/ml immiquimod) in the presence or absence of 15 μg/ml PspA for 24h. For some experiments, DCs were incubated with biopharmacological inhibitors to various molecules. For example, DCs were incubated with 100 μM TMB-8 for inhibiting calcium release from intracellular stores, 3 mM EGTA for inhibiting calcium influx from external medium, 10 μM U0126 for inhibiting MAPK-ERK and 10 μM diphenyleneiodonium (DPI) for inhibiting reactive oxygen species (ROS). For some experiments, DCs were transfected with siRNAs to various genes as described before prior to stimulation with 15 μg/ml PspA [6/ml) were transfected with 60 pmol of siRNA for 72h using the Hiperfect transfection reagent  in OPTIMEM medium . GM-CSF was added 5h following transfection, and incubation was continued for 72h for DC differentiation. Knockdown was verified by semi-quantitative reverse transcription-polymerase chain reaction. Six to eight week old female BALB/c mice reared in a pathogen-free environment were used throughout the study. DCs were differentiated from mouse bone marrow as described previously . Brieflyd before . The purAt the end of the incubation period, DCs were first incubated with Fc-Block  to mask CD16/CD32 Fc receptors and stained for the surface expression level of PD-L1 using phycoerythrin-tagged monoclonal antibody to PD-L1 and analyzed by flow cytometry  as described previously , 40. The6/ml) were incubated with recombinant PspA3–286 from R36A [DCs , Mitogen Activated Protein Kinase-Extracellular-signal Regulated Kinase (MAPK-ERK), PhosphatidylInositide-3 Kinase (PI-3K), Protein Kinase A (PKA), Calcium/calmodulin dependent protein Kinase II α (CAMKIIa) during PspA induced PD-L1 expression. To inhibit calcium homeostasis, we treated DCs with EGTA to chelate extracellular calcium and TMB-8 to inhibit intracellular calcium release from the endoplasmic reticulum. Interestingly, inhibiting intracellular calcium burst completely inhibited PspA induced PD-L1 expression, while inhibiting calcium influx from external milieu further increased PspA induced PD-L1 expression . This inM. tuberculosis infection [Inhibiting ERK-MAPK with U0126 increased PspA induced PD-L1 expression suggesting the involvement of this pathway in PD-L1 expression . On the nfection . As show3R with TMB-8 completely abrogated PspA induced PD-L1 expression, while inhibiting calcium influx from extracellular medium using EGTA increased PD-L1 expression  proteins STIM1 and STIM2, the molecular sensors that regulate Store Operated Calcium Entry (SOCE) via the Calcium Release Calcium Activated Channel (CRAC) ORAI1. As shown in the S. pneumoniae. To that end we incubated DCs with wild type D39, strain R6 (derived from D39 that lacks capsule but has PspA) or JY2008 (lacks capsule and surface PspA). As shown in S. pneumoniae infection.We next investigated whether the effects observed with PspA would be replicated during infection with 3R) and EGTA  followed by incubation with S. pneumoniae D39 and PD-L1 expression was monitored. Similar to the data obtained with PspA Click here for additional data file.S2 FigMouse bone marrow derived DCs were transfected with siRNAs directed against the indicated gene for 36h as described previously . Total R(DOC)Click here for additional data file.S3 FigMouse bone marrow derived DCs were stimulated with 15 μg/ml PspA for 24h and PD-L1 expression was monitored by flow cytometry. Thick line represents PspA stimulated cells stained with isotype matched control monoclonal antibody. Dotted line represents unstimulated cells stained for PD-L1. Thin line represents PspA stimulated cells stained with antibody specific to PD-L1.(DOC)Click here for additional data file.S4 FigMouse bone marrow derived DCs were transfected with siRNA against TLR2 for 36h, followed by stimulation with PspA for 24h. PD-L1 levels were monitored using flow cytometry. In Panel A, dotted line represents unstimulated cells transfected with control siRNA. Thin lines represent cells transfected with control siRNA followed by stimulation with PspA for 24h. Bold line represents the cells transfected with siRNA specific to TLR2 followed by stimulations with PspA. Data from one of three independent experiments is shown. Panel B, shows the relative expression of PD-L1 represented as relative MFIs. Panel C shows the knockdown efficiency of siRNA against TLR2 in DCs.(DOC)Click here for additional data file.S5 FigMouse bone marrow derived DCs were stimulated with PspA for 2h. 30 minutes prior to the incubation period cells were loaded with 10 μM DCFH-DA. At the end of incubation period, cells were quickly and thoroughly washed with culture medium and immediately analyzed for ROS level by flow cytometry. Thin line represents unstimulated cells stained for ROS. Bold line represents cells stimulated with PspA (15 μg/ml).(DOC)Click here for additional data file.


Digital game-based learning environments provide emerging opportunities to overcome learning barriers by combining newly developed technologies and traditional game design. This study proposes a quantitative research approach supported by expert validation interviews to designing a game-based learning framework. The goal is to improve the learning experience and decision-making skills of soccer referees in Turkey. A serious game was developed and tested on a group of referees (N = 54). The assessment results of these referees were compared with two sample t-test and the Wilcoxon signed-ranked test for both the experimental group and the control group. The findings of the current study confirmed that a game-based learning environment has greater merit over the paper-based alternatives. Football is the world’s most popular sport with approximately 33.4 million individuals involved   Laws of the Game (LOG) book consists of the rules of the game of football  was essential where it was important to assess their requirements and monitor their progress. In addition, it was important to include a qualitative aspect in this study as the viewpoints of referees were crucial to address the design challenges during the development of such an interactive software-intensive system.For the qualitative part of the study, expert reviews were conducted on five experienced referees who had been working in the education department of the Turkish Football Federation for at least 10 years when a database of 700 questions for the game platform was prepared. The protocol used for the expert reviews were based on the following questions “Do you think this question is suitable for assessing a novice referee?”, “Can you please rate this question between 1 and 3 in terms of its difficulty level?”, “What would be learning outcome for this question?”, “Can the referees reach this question in the other platforms?” and “Is this question similar to the questions that are used in your training programs?”.Q1: Do you think you can improve your knowledge level and decision-making skills by playing the game?Q2: Which question category/categories are more useful for you?Q3: Do you prefer to play the game or study from the FIFA book?Q4: What kind of features do you think are missing in the training framework?Q5: Do you like to have a competition between the other players? If your answer is yes, is it useful or not?For the final qualitative phase of the study, validation interviews were conducted with the participants so as to explore the benefits and potential issues encountered during the interactive assessments by asking these questions:The answers were evaluated and categorized as positive, negative or neutral by the five retired first class football referees (who had managed matches in the Super League for at least 7 years).For the quantitative part of the study, an interactive assessment (i.e. pre-test and post-test) was conducted in the form of a game-like structure to a group of football referees, namely those who have joined the referee training program. The aim was to explore the benefits of the proposed platform in improving the knowledge level and decision-making skills of these football referees by measuring the differences between the referees’ pre-test and post-test scores. The numerical results of both tests were analyzed by using statistical techniques to understand whether or not a significant difference between the groups exists.A serious game is a special type of game which is used to educate (or sometimes inform) participants about a selected topic Abt . Here, aTrue/False Questions and Multiple Choice Questions categories are selected from the actual questions that are administered every year to measure a referee’s level of knowledge of the rules of football while the questions in the Video Questions category consist of videos of critical positions that occur in real matches that were played during past football seasons.The game has three different types of questions related to the laws of the game of football, which are: (1) True/False Questions, (2) Multiple Choice Questions and (3) Video Questions. The questions in the In addition to the question categories, a virtual game board was designed. The game board consists of a set of steps as shown in Fig. Using a traditional perspective, the participants use a virtual dice with three faces which are corresponding with three numbers. Each number on the die represents each question category as follows: (1) represents True/False Questions, (2) represents Multiple Choice Questions, and (3) represents Video Questions.First, the player throws the die, and produces a one, two or three. Based on the number thrown, he takes a question from one of the corresponding categories above. For example, if the die shows 3, the player takes a question from the video questions category.If the player answers correctly to the question, he moves one step forward. Otherwise, he moves one step backward (unless the player is at the starting point or at a bonus step). Before going one step backward, the correct answer to the questions and a detailed explanation of the correct answer is shown to the player.As a simple reward structure, there is a group of checkpoints in the game known as bonus steps. The bonus steps are designed to keep the player in the game. This also prevents players from answering incorrectly to a group of questions and returning to the starting point. In addition, this mechanism incrementally provides the player with small rewards thereby achieving motivation.The bonus steps also offer additional time for correct answers to a question being asked in this step. The first bonus step gives 5 s of bonus time, the second bonus step offers 10 s, the third bonus step offers 15 s, and finally 20 s are added as bonus time for the fourth bonus step.The timer of the game starts with the first question and it stops when a player answers the final question.Finally, each player’s racecourse completion time is compared and placed in a table which sorts the players according to their completion time. The player at the top of the leader-board is the winner. The flow of the game is illustrated in Fig. The mechanics of the game can be identified as follows:To evaluate the effect of our game correctly, we conducted experiments in which we allowed participants play the game. Following this, we analyzed the results of these experiments. Creating a sample is necessary in order to obtain valid results. The members of the sample group are selected to have a similar level of knowledge at the beginning of the training program. In order to ensure this similarity, 54 football referees, all of whom had completed the referee course at the same time, were selected. These referees were randomly separated into a control group (N = 27) and an experimental group (N = 27). The referees in the control group are not allowed to log in to our game system. They were only able to learn the rules of the game from the LOG book. The referees in the experimental group were allowed to log in to our game system. However, they were not permitted to use LOG book.Multiple Choice QuestionsVideo QuestionsIn order to evaluate the level of knowledge of the referees regarding the game rules, a pre-test was administered to a selected group of referees for the training program at the beginning of the training period. This test consisted of 50 questions, which were separated into two sections thus:From a group of questions, 35 multiple choice questions and 15 video questions were selected.To build a proper assessment tool, an expert from the Turkish Football Federation was interviewed. The number and content of questions in the test was determined by him. This expert indicated that there are 50 questions in the real exam administered by the Turkish Football Federation to measure a referee’s level of knowledge of the rules of the game. Hence, the number of questions determined in both tests was 50.The pre-test was administered on 9 December 2014, at which there were 54 football referees as participants. The pre-test results of both groups are shown in Fig. Important points were considered during the data analysis. The first important point is that the average scores of both groups were very near to each other in every part of the exam. In other words, in both groups, the referees’ level of knowledge of the game was nearly identical at the beginning of the training program. The second important point was the number of successful referees in the exam. In real exams, 70 out of 100 is the threshold for referees to be successful. When the pre-test results were examined, from 27 individuals, 4 were found who were able to pass the exam in the experimental group .To improve the quality of training, further research should also be conducted to provide a more immersive environment with computer-generated landscapes with a 3D virtual soccer field that could be based on virtual reality or augmented reality. Such a system would provide full simulation of a match in a virtual football field, which can provide the trainee a real life like a learning experience. A further study with more focus on referee training on such a medium that is based on the tracking of the positions and actions of a participant with continuous feedback on an artificial interactive computer-generated football field is therefore suggested.


Cases with IHC score of 1, 2, 3 all underwent apparent decrease (p = 0.007). However, no significant changes were observed on tumour-infiltrating immune cells (ICs) (p = 0.337). Subgroup and semiquantitative analyses all presented similar results. Moreover, patients with response to NACT presented significantly reduced PD-L1 expression on TCs (p = 0.004). Although it was not confirmed by the Cox proportional hazard regression model, there was an apparent difference in disease-free-survival (DFS) between negative-to-positive switch of PD-L1 status and the contrary group . Our data revealed that antecedent chemotherapy for NSCLC may results in inconsistency of PD-L1 expression. PD-L1 expression is suggested to be monitored around treatment and on serial samples, at least, on the latest tumor specimen.The effects of treatments to programmed death ligand-1 (PD-L1) expression is unknown. The aim of this study was to investigate the impact of neoadjuvant chemotherapy (NACT) on PD-L1 expression in non-small cell lung cancer (NSCLC) patients. PD-L1 expression was detected by immunohistochemistry (IHC) method in 32 paired tumor specimens pre and post-NACT. The positivity of PD-L1 on tumor cells (TCs) changed from 75% to 37.5% after NACT ( Global cancer statistics indicate that lung cancer remains the leading cause of cancer-related mortality2Recent insights into the molecular mechanisms governing the host response to cancer cells has led to the identification of important signaling pathways and checkpoint molecules involved in the anticancer immune response456789101012131012141516181920222425Based on above data, the prognostic and predictive value of PD-L1 IHC was assumed to temporally dependent on the antecedent treatment and time of biopsy. We therefore conducted this exploratory analyses with paired NSCLC specimen pre and post-NACT to explore the impact of chemotherapy on PD-L1 expression. The association between PD-L1 change patterns and prognosis were also analyzed.2, days 1 and 8 plus cisplatin 75 mg/m2, days 1, every 3 weeks). The median tumor size at baseline was 35.0 mm (IQR: 26.3–40.0). After the NACT, the median value decreased to 22.0 (IQR: 18.0–30.3). This change was significant according to the result of Wilcoxon test (p < 0.001). Finally, partial response were found in 18 (56.2%) patients, while 50% patients (n = 16) achieved down-staging after NACT.Finally, 32 patients  were included for analyses. Baseline demographics and PD-L1 expression, as presented by H-score, are summarized in p = 0.003). However, a contrary result was found in ICs, whose PD-L1 positive rate increased from 43.8% to 56.2% after NACT, although no statistical significance was reached (p = 0.319).p = 0.007). Subgroup analyses also supported the finding, as summarized in p = 0.337).Another perspective is, as shown in p = 0.005, p = 0.474, p = 0.004, p = 0.378, p = 0.441, 1.000 separately, Semiquantitative analyses demonstrated that the H-score of PD-L1 on TCs was significantly decreased . Besides, no significant differences were detected among four change patterns of PD-L1 on ICs  months. As shown in The PD-1/PD-L1 axis is one of the crucial mechanisms underlying immune escape of tumor cells. These pathways are currently attractive therapeutic targets for human cancers, including NSCLC. One of the provocative findings accompanied with anti-PD-L1/PD-1 exciting efficacy involves the potential predictive value of PD-L1 expression on tumor cells27Our result demonstrated that the expression of PD-L1 on tumor cells is not consistent for patients with NSCLC. The expression profile was correlated with antecedent chemotherapy. Therefore, the dynamic property of PD-L1 expression demonstrated by our study may provide a possible reason to why negative status from chemo-naive sample finally confer certain responses when PD-1/PD-L1-directed therapy are given in second or later lineset al. reported that cytotoxic agents, specifically paclitaxel, etoposide and 5-fluorouracil, could induce PD-L1 surface expression in breast cancer cells, which lead to promoted PD-L1-mediated T cell apoptosiset al. have revealed doxorubicin-dependent down-regulation of cell surface PD-L1p = 0.004) on TCs, but not in those not responded (p = 0.196). To verify the hypothesis, we analyzed the difference of PD-L1 expression on tumor-infiltrating lymphocytes (ICs). No significant alteration was observed, neither in the response group nor non-response group. Of note, whether drug resistance induces PD-L1 expression is largely unknown. In present study, no significant induction of PD-L1 on tumor cells was detected in population exerted no response to NACT. We thereby proposed non-monotonic mechanisms underlying the effect of chemotherapy on PD-L1 expression, including both cytotoxic-related PD-L1 reduction and other signal-associated PD-L1 regulation. It has been identified that constitutive IFN-gamma secretion or oncogene activation will stimulate PD-L1 expression. Besides, PD-L1 up-regulation mediated by IFN-gamma is commonly considered as a classically adaptive way for immune resistance31Many anticancer agents exert immunomodulatory effects on host system in addition to their cytotoxicityet al. reviewed that PD-L1 and PD-L2 did not correlate to the prognosis of NSCLC, nor did they relate to other clinically pathological factorset al. reported that high expression of PD-L1 in the primary foci of NSCLC was an independent predictor of poor prognosiset al. revealed that high PD-L1 expression is independently associated with longer overall survival and correlated with high tumor grade and younger patient ageThe prognostic value of PD-L1 has also been well-discussed among various cancers. PD-L1 expression on tumor cells correlates with poor clinical prognosis of renal, ovarian cancers, breast cancers151617For patient with pathological N2 NSCLC, the most menacing problem is postoperative recurrence. Thus new therapeutic targets for NSCLC are urgently needed. Alternative strategies such as immunotherapy or combination strategies involved of immunotherapy are now being considered for NSCLC treatment. CheckMate 012 investigate the efficacy and safety of combining anti-PD-1 antibody nivolumab with platinum doublet (n = 56), which achieved ORR as 33–50%, and remarkable 1-year survival rate as 59–87%This study is limited by retrospective feature and relatively small samples. Althouth we present an interesting and relevant finding, further research are warranted to explore the intrinsic mechanism of chemotherapy-induced alteration of PD-L1 expression. Considering the complexity of tumor micro-environment, deep illustration about effects of chemotherapy on tumor infiltrating lymphocytes patterns may provide better interpretation. The alteration of PD-L1 expression confers potentially impact on future treatment decision. Despite certain limitations, our data was the first to demonstrate the inconsistance of PD-L1 around chemotherapy and highlight the need to monitor PD-L1 biological changes around treatment and on serial samples, at least, the latest biopsy.Based on our result, the expression of PD-L1 on tumor cells is not consistent around chemotherapy for patients with NSCLC. The value as a predictive biomarker for anti-PD-1 efficacy and future immunotherapeutic decision should be cautious about the inconstancy of PD-L1. PD-L1 expression is suggested to be monitored around treatment and on serial samples, at least, on the latest tumor specimen.All patients were evaluated in a multidisciplinary meeting for treatment strategy setting. Patients pathologically diagnosed with NSCLC and received NACT prior to surgical resection at Sun Yat-sen University Cancer Center  from January 2010 to March 2014 were screened for eligibility. We only included cases met all of the following conditions: 1). diagnosed as stage IIIA (N2)/IIIB (T4N0-1) by invasive mediastinoscope; 2). received 1-4 cycles neoadjuvant chemotherapy before radical resection; 3). with enough paired tumor tissue samples around NACT for IHC staining of PD-L1.This retrospective study was approved by the Institutional Review Board of Sun Yat-sen University Cancer Center. All the patients had provided written informed consent before tissue samples were collected. All procedures performed in studies involving human participants were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The studies conducted in laboratory were operated under exploratory research principles.2 IV, days 1) plus carboplatin  or cisplatin , every 3 weeks. 2. Pemetrexed-based NACT, including pemetrexed  plus cisplatin , every 3 weeks. 3. TKI-based NACT, means treatment including erlotinib , gefitinib  or icotinib  for a period of at least 4 weeks, stopped 72 hours before surgery. All patients were re-evaluated by radiographic method after two cycles of treatment (four weeks for TKI-based NACT) for resectability. Adjuvant chemotherapy was administrated rouinely after resection, according to the sensitivity to neoadjuvant regimen and pathological type of cancer.Patient diagnosed as stage IIIA (N2)/IIIB (T4N0-1) by invasive mediastinoscope were transferred to medical oncology department for neoadjuvant chemotherapy. According to the regimens given to patients, the neoadjuvant chemotherapy (NACT) were classified as three types: 1. Paclitaxel-based NACT, which refers to paclitaxel , EGFR mutation status and classification of neoadjuvant regimens.The expression of PD-L1 in human NSCLC specimens was performed with IHC staining using rabbit monoclonal anti-human antibody . Five-μm-thick Sections were cut from the formalin-fixed, paraffin-embedded (FFPE) tumor block and then routinely deparaffined and rehydrated. For antigen retrieval, slides were heated in a microwave oven for 30 minutes in citrate buffer solution (pH = 7.4) and cooled slowly at room temperature for 20 minutes. Then, we blocked the activity of endogenous peroxidase with 3% hydrogen peroxide for 8 minutes. Thereafter, the sections were treated with primary antibodies and incubated for overnight (more than 12 hours). Subsequently, the slides were rinsed in PBS three times and incubated in HRR-linked secondary antibodies. After incubation, slides were washed again with PBS and then visualized using diaminobenzidine. Finally, Mayer’s hematoxylin was used to counterstain the sections and dehydrated and mounted. This mature method was applied in our previous work and detailed describedSemiquantitative H score  was defined as multiplying the percentage of stained cells by an intensity score 19p-value of <0.05 was considered statistically significant at all situation.All the statistical analysis was performed using SPSS 20.0 for Windows . Non-parametric quantitative data were presented as median value and interquartile range (IQR). Wilcoxon or Mann-Whitney method was used for comparision of non-parametric data. Pearson’s chi-squared test or Fisher exact test was used to assess the correlation between changes of PD-L1 expression and clinicopathologic variables. DFS was defined as the time from data of surgery to recurrence. Survival analyses were performed by Kaplan-Meier method and Cox proportional hazard regression model. A two sided How to cite this article: Sheng, J. et al. Expression of programmed death ligand-1 on tumor cells varies pre and post chemotherapy in non-small cell lung cancer. Sci. Rep.6, 20090; doi: 10.1038/srep20090 (2016).