Quenching thermoelectric effects in graphene leads to an extraordinary increase of the linear dynamic range in photodetectors. 3-intercalated graphene using laser irradiation. Photocurrent measured at these planar junctions shows an extraordinary linear response with an LDR value at least 4500 times larger than that of other graphene devices (44 dB) while maintaining high stability against environmental contamination without the need for encapsulation. The observed photoresponse is purely photovoltaic, demonstrating complete quenching of hot-carrier effects. These results pave the way toward the design of ultrathin photodetectors with unprecedented LDR for high-definition imaging and sensing.Graphene-based photodetectors have demonstrated mechanical flexibility, large operating bandwidth, and broadband spectral response. However, their linear dynamic range (LDR) is limited by graphene’s intrinsic hot-carrier dynamics, which causes deviation from a linear photoresponse at low incident powers. At the same time, multiplication of hot carriers causes the photoactive region to be smeared over distances of a few micrometers, limiting the use of graphene in high-resolution applications. We present a novel method for engineering photoactive junctions in FeCl Intense research activity on graphene-based photodetectors (3 has been found to result in record-high levels of hole-doping (≈ 1 × 1015 cm−2) with a room temperature electrical conductivity up to 1000 times larger than that of pristine graphene while maintaining equivalent absorption over the visible wavelength range  response and an LDR as large as 44 dB, which is at least 4500 times larger than that of any previously reported graphene photodetector  defines a new paradigm in ultrathin, high-LDR photodetectors.The light-assisted design of integrated and atomically thin optoelectronic circuits is a step forward to a new frontier in high-definition sensing applications, whereas FeCl3 introduced only between the top three carbon layers. Intercalation of FeCl3 molecules into mechanically exfoliated FLG on a Si/SiO2 substrate was conducted using a previously reported method (0 peak at 1580 cm−1 due to the E2g phonon mode of pristine graphene as well as the blue-shifted G1 = 1615 cm−1 and G2 = 1625 cm−1 peaks of the same mode caused by the charge doping of FeCl3 molecules adjacent to only one side of a graphene layer (stage 2) or sandwiching the carbon atoms (stage 1) .The starting material to achieve our goal is an intercalated four-layer graphene flake with FeCle 1) see . Upon itpeak see . All the3 intercalation in graphene, we exposed a 5.5-μm-wide section of the intercalated flake to a raster laser scan (15.3 MW/cm2 for 3 s in 0.5-μm steps). Raman spectra were collected at incrementally spaced locations across the laser-exposed region both before and after illumination, as shown in 1 and G2 peaks along a 21-μm line scan. Uniform removal of the G2 peak from the entire rastered region demonstrates that FeCl3 molecules may be displaced from arbitrarily mapped areas. The degree of intercalation remains unchanged away from the irradiated area, with the resolution of FeCl3 displacement defined by the laser spot profile. The marked effectiveness of laser-induced deintercalation over a significant fraction of the FeCl3-FLG flake area presents an elegant method, akin to optical lithography, which can be used to locally customize the chemical functionalization of graphene layers.To ascertain the effectiveness of laser irradiation as a method for locally tailoring FeClet al. , across a graphene junction formed by regions with a differing density of states. If the junction is illuminated, a local increase of temperature (ΔT) results in the diffusion of carriers and an opposing photovoltage (VPTE = ΔS ΔT) is generated. Hot-carrier dynamics are generally recognized to dominate photocurrent generation in supported graphene devices because of inefficient cooling of electrons with the lattice (nph) of carriers, which, in the presence of an in-built electric field, are separated and induce current at the electrodes  generated in one of several measured monolayer graphene devices (section S2.4), where α = 2/3 was obtained with 10 mV applied between the source and drain. On the other hand, the photoresponse of FeCl3-FLG is strikingly different from that of pristine graphene, exhibiting a linear dependence extending beyond three logarithmic decades of incident laser power. The observed difference originates from the charge carrier dynamics. More specifically, in pristine graphene, the chemical potential (μ) lies close to the charge neutrality point, and the small Fermi surface imposes tight constraints on the maximum energy lost through momentum-conserving acoustic phonon emission  (Th >> Tl) and are instead cooled via short-range “supercollision” processes at sites of disorder  to justify the observed linear power dependence (Th can be explained by the ultrahigh levels of charge density (up to 3 × 1014 cm−2 per layer) achieved through FeCl3 intercalation  illuminated by a laser (spot diameter l0) isR is the resistance of the graphene layer and η is the carrier mobility. For a doped graphene layer with a charge carrier density above n ≈ 3 × 1013 cm−2, the Bloch-Grüneisen temperature (TBG = ΔEac/kB) exceeds 300 K  from several micrometers (L/2. Using the low-energy density of states for monolayer graphene and a minimum conductivity of σmin ≈ 4e2/h (min(1 + μ2/Λ2), where Λ ≈ 140 meV. The Mott relation for thermopower (IPTE/IPVE ≈ − 0.06 using 3-FLG p-p′ junctions and acts in the opposite direction to dominant photovoltaic processes. Opposing photocurrents at p-p′ junctions have previously been predicted in monolayer graphene transistors with split electrostatic gates (S ∝ −σ−1 (dσ/dμ)), the density of states of graphene dictates that these gradients will always point in opposite directions so long as the chemical potentials on each side of a photoactive junction are both situated in the valence band (p-p′ junctions) or in the conduction band (n-n′ junctions) away from the charge neutrality point. As a result of these findings, we take the direction of photocurrent signals shown in 3-FLG detectors.To identify the origin of photocurrent at p-p′ junctions of FeCl3-FLG–based photodetectors would not require thermal isolation and could be packed to a far higher density than undoped graphene monolayers, making them well-suited for imaging applications over a broad spectral range.Through chemical functionalization, the selective quenching of thermoelectric processes in graphene could prove to be a highly useful tool for extending the use of graphene-based light sensors beyond microbolometers and modulators suitable for infrared wavelengths. Pixels of FeCl3-FLG detectors is characterized by an extraordinary LDR. The noise-equivalent power (NEP) of our device was measured to be 4 kW/cm2 (see section S2.2), thus resulting in an LDR of 44 dB. This is 4500 times larger than that of previously reported graphene photodetectors (LDR ≈ 7.5 dB)  to red (685 nm). Fits of the power exponent at each wavelength give the following: α375 = 0.99 ± 0.01, α473 = 1.05 ± 0.06, α514 = 0.97 ± 0.03, α561 = 0.99 ± 0.01, and α685 = 0.95 ± 0.05. For the multitude of FeCl3-FLG devices measured, we observed no deviation from a strictly linear power dependence in the whole measured power range. This indicates that the ultrahigh degree of charge carrier doping introduced by FeCl3 intercalation acts as a unique stable method for quenching thermoelectric effects and fixing the photoresponse to an extended linear dynamic regime, avoiding the sensitivity to processing methods and environmental conditions, which pristine graphene photodetectors (Iph/Popt(λ), of a p-p′ junction is displayed with and without correcting for reflections from the Si/SiO2 substrate (section S6). The photoresponse remains markedly consistent across the entire visible range, where ℜ(λ) varies by only one order of magnitude, with values >0.1 mA/W, which are typical for high-end all-graphene photodetectors , where NA is the numerical aperture of the objective]. To explore the density to which graphene-based imaging pixels may be packed in the absence of hot-carrier effects, we use scattering-type near-field optical microscopy  to define photoactive junctions below the Abbe limit. This technique has been used extensively to study the plasmonic –grown graphene has already been demonstrated . Raman spectra were acquired with a Renishaw spectrometer equipped with a 532-nm laser focused to a 1.0-μm spot through a 50× objective lens. An incident power of 1 mW was used for all measurements, and spectra were recorded with a 2400 g/mm grating. A charge-coupled device acquisition time of 5 s was used.Raman spectroscopy measurements used to characterize the degree of intercalation in FeClT = 300 K, P = 1 atm) in air. The laser power was varied from 1.5 μW to 1 mW by analog modulation of the laser diodes and the use of neutral density filters along the beam path. All the devices studied have been measured in air over a time scale longer than 1 year, during which no change in the photoresponse was observed.A continuous-wave laser beam from a multiwavelength  solid-state laser diode array was focused onto the sample through a 50× lens, producing a spot size of 1.0 μm. A high-resolution microscope stage (minimum step size of 0.05 μm) was used to produce spatial maps of the photocurrent. Electrical measurements were performed in short-circuit (zero bias) configuration using a DL Instruments Model 1211 current amplifier connected to a Signal Recovery model 7124 digital signal processing lock-in amplifier. The lasers were modulated at a frequency of 73.3 Hz with a transistor-transistor logic signal from a direct digital synthesizer function generator, which was used as a reference signal for the lock-in. All measurements were performed under ambient conditions  tip, which creates a strong, exponentially decaying field at its apex. The tip is then scanned across the sample, operating in tapping mode, allowing parameters, including topography and scattered light emission, to be measured with subwavelength resolution (


Lung adenocarcinoma (AD) is a common variant of non-small cell lung cancer (NSCLC). Programmed cell death protein 1/programmed cell death ligand 1 (PD1/PD-L1) are promising immunotherapy targets and its expression may be an important biomarker of predicting clinical response. In this study, we evaluated PD-L1 expression in conjunction with clinicopathological characteristics and outcomes in resected lung adenocarcinoma.P = 0.219, highly correlated). PD-L1 was highly expressed in male patients and smokers with lung adenocarcinoma , while no associations were identified between PD-L1 expression and age, tumor size, clinical stage, positive pleural invasion, lymph node metastasis, or therapy methods. Overexpression of PD-L1 was a significant indicator of shorter recurrence free survival time and overall survival . Multivariate analysis revealed that PD-L1 expression was an independent risk factor for poor recurrence free survival and overall survival .This study included 133 cases of lung adenocarcinoma. PD-L1 expression rate in lung adenocarcinoma was 16.5% at the mRNA level and 13.5% at the protein level, and the kappa coefficient of the two examination methods was 0.824  rabbit monoclonal antibody. mRNA levels of PD-L1 were evaluated using PD-L1 overexpression is more frequently observed in male patients and smokers in lung adenocarcinoma. PD-L1 expression is an indicator of worse prognosis in surgically resected lung adenocarcinoma patients. Lung adenocarcinoma is a common variant of non-small cell lung cancer (NSCLC). In recent years, there have been dramatic advances in the treatment of lung adenocarcinoma because of the development of therapies targeting driver oncogene alterations, for example, drugs targeting the epidermal growth factor receptor (EGFR) mutation or the anaplastic lymphoma kinase (ALK) fusion , 2. HoweImmune checkpoints are inhibitory pathways that can maintain self-tolerance and protect peripheral tissues by modulating immune response . Overexpin situ hybridization at the mRNA level. Furthermore, we compared the expression of PD-L1 with clinicopathological characteristics and outcomes in lung adenocarcinoma.In this study, we evaluated PD-L1 expression using immunohistochemistry at the protein level and by The clinicopathological characteristics of the lung adenocarcinoma patients are summarized in Table The growth pattern of lung adenocarcinoma was classified into lepidic (16 tumors), acinar (65), mucinous (8), solid (18), papillary (19), and micropapillary (7) patterns. Pleural involvement was identified in 92 cases. Positive node metastasis was present in 53 cases.P = 0.219).Of the 133 cases of lung adenocarcinoma examined in this study, the PD-L1 expression rate in lung adenocarcinoma detected by IHC and ISH was 13.5% (18/133) and 16.5% 22/133), respectively. The two techniques were consistent in identifying 110 cases as PD-L1 negative, and 17 cases as PD-L1 positive. Representative cases of IHC and ISH results are shown in Figure 2/133, reP = 0.019); in smokers than non-smokers (P = 0.002); and in solid, papillary, or micropapillary growth pattern tumors compared to acinar and lepidic growth pattern tumors (P = 0.000). No significant association was detected between expression of PD-L1 and patient age , tumor size , clinical stage , pleural involvement (P = 0.553), or lymph node metastasis (P = 0.439).Expression of PD-L1 was significantly higher in male patients than in female patients (P = 0.000) and OS (P = 0.000)  and overall survival (OS) times were 32.00 and 34.70 months, respectively. Forty-eight patients experienced recurrence at a median follow-up time of 14.00 months. Twenty-one patients died at a median follow up time of 22.60 months. Kaplan–Meier analysis revealed that PD-L1 expression was significantly associated with a shorter RFS . A recent study compared and validated 6 commercially available PD-L1 monoclonal antibodies . Their results showed that all 6 antibodies had high levels of concordance (R2 = 0.76–0.99). They further suggested that previously described differences in PD-L1 expression in tissue are independent of the antibody used and likely attributable to tumor heterogeneity, assay- or platform-specific variables, or other factors [Evaluating human epidermal growth factor receptor-2 in breast cancer by IHC, which is economic and time-saving, has been successful. Efforts are being made to establish a method to evaluate PD-L1 expression by IHC. A multitude of detection PD-L1 IHC antibodies have been utilized, including the Ventana SP263 and SP142 clones, and the Dako 28–8 and 22C3 clones –13. VariThe PD-L1 expression rate in lung adenocarcinoma samples in our study was about 13%. Previously reported expression rates of PD-L1 using different IHC antibodies range from 8% to 55% in lung adenocarcinoma or NSCLC patients –18. ThisIn our study, we observed significant correlation of PD-L1 expression with clinicopathological characteristics. PD-L1 expression is associated with smoking habits. Smoking exposure may induce inflammatory as well as suppressive effects on the immune system . Others Expression of PD-L1 was reported to be correlated with poor clinical outcomes in a number of human cancers, including lung, melanoma, breast, bladder, ovarian, pancreatic, esophageal adenocarcinoma, and kidney tumors . In our In conclusion, we have demonstrated that expression of PD-L1 was present in 13.5% of lung adenocarcinoma patients and was associated with a poorer prognosis. Further studies are warranted to confirm PD-L1 expression, and the therapeutic effect of PD-1/PD-L1 blockade, in clinical trials.One hundred and thirty-three patients were enrolled in this study, and underwent a surgical operation with a final diagnosis of lung adenocarcinoma at Peking Union Medical College Hospital  between January 2012 and December 2015. For poorly differentiated cases, additional immunostaining for Napsin A and thyroid transcription factor 1 (TTF-1) was performed to confirm the final results.th, 2016.Clinical information for the patients was retrieved from the PUMCH digital database, including age, gender, smoking habit, clinical stage, treatment methods, RFS, and OS. Clinical stage was determined at the time of surgery, according to the American Joint Commission on Cancer (AJCC), 7th edition, tumor-node-metastasis staging system. RFS was defined as the time from operation to relapse or until the endpoint of the study. OS was calculated as the time from surgery to death or until the endpoint of the research, which was April 30For each tumor sample, hematoxylin-eosin staining (HE) slides were all re-reviewed by two experienced pathologists to confirm the final diagnosis. The growth pattern of lung adenocarcinoma was defined according to the guidelines of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society classification system that includes lepidic, acinar, papillary, micropapillary, and solid predominant patterns. Both pathologists had no knowledge of the clinical information or PD-L1 expression status of these cases during the review. Additionally, the following pathological characteristics were evaluated: tumor size, pleural invasion, and node metastasis status.This study was approved by the Institutional Review Board of Peking Union Medical College Hospital. Informed consent was obtained from all patients before they underwent the operation.The selected area of representative morphology was labeled on the HE slides. The corresponding formalin-fixed paraffin-embedded (FFPE) primary tumor specimens were obtained from the Department of Pathology. A tissue microarray (TMA) construction machine  was used. Three core-tissue biopsies, 1.0 mm in diameter, were collected for each case.The IHC study was performed on formalin-fixed, paraffin-embedded TMA sections, with a thickness of 4 μm, using standard autostaining protocols on a Ventana Benchmark ULTRA autostainer . The PD-L1 antibody used in our study was SP263 . Tumors with positive membrane staining in more than 25% of the whole tumor were deemed positive, as suggested in a study by Ratcliffe et al. .in situ hybridization (ISH). An RNAscope FFPE 2.0 HD detection kit  was used according to the manufacturer’s instructions. Briefly, 5 μm-sections were deparaffinized, boiled with pre-amplification reagent for 15 minutes and submitted to protease digestion followed by hybridization for 2 h with a mixture containing target probes against human PD-L1, ubiquitin C (UBC) as a positive control, and the bacterial gene DapB as a negative control. Hybridization signals were detected with 3,3′-diaminobenzidine. Positive staining was indicated by brown punctate dots in the cytoplasm or nucleus. PD-L1 mRNA expression levels were categorized into 5 grades according to the manufacture’s scoring guideline: score 0, no staining or < 1 dot per cell; score 1, 1–3 dots per cell (visible at 20–40X); score 2, 4–10 dots per cell (visible at 20–40X); score 3, > 10 dots per cell and < 10% positive cells have dot clusters (visible at 20–40X); score 4, > 10 dots per cell and > 10% positive cells have dot clusters (visible at 20–40X). We grouped score 0, 1, 2 as negative, 3 and 4 as positive.For detection of PD-L1 expression, at the mRNA level, we used The relationship between PD-L1 expression and clinicopathological characteristics was analyzed using the chi-squared test. Survival analysis was calculated by the Kaplan-Meier method, the log rank test was used to compare the difference of two groups, and multivariate analysis using the Cox regression model was conducted to analyze the related clinicopathological features. All tests were 2-sided. P < 0.05 was considered statistically significant. The statistical analyses were performed using SPSS software .κ-value was considered as follows: value ≤ 0.40, poor to fair concordance; 0.41–0.60, moderate concordance; 0.61–0.80, substantial concordance; 0.81–1.00, almost perfect concordance.For analysis of concordance between the results of IHC and RNA ISH, the kappa value was calculated. The significance of the


Q) factor to ~18.5 from ~3.23 at room temperature and normal pressure, the limited effects of air damping on resonance behaviors at 10−2 and 105 Pa were demonstrated by characterizing graphene F-P resonators with open and micro-air-gap cavities. Then in terms of optomechanical behaviors of the resonator with an air micro-cavity configuration using a polished ferrule substrate, measured resonance frequencies were increased to the range of 509–542 kHz from several kHz with a maximum Q factor of 16.6 despite the lower Knudsen number ranging from 0.0002 to 0.0006 in damping air over a relative pressure range of 0–199 kPa. However, there was the little dependence of Q on resonance frequency. Note that compared with the inferior F-P cavity length response to applied pressures due to interfacial air leakage, the developed F-P resonator exhibited a consistent fitted pressure sensitivity of 1.18 × 105 kHz3/kPa with a good linearity error of 5.16% in the tested range. These measurements shed light on the pre-stress-dominated pressure-sensitive mechanisms behind air damping in in situ F-P resonant sensors using graphene or other 2D nanomaterials.We demonstrated a miniature and in situ ~13-layer graphene nanomechanical resonator by utilizing a simple optical fiber Fabry-Perot (F-P) interferometric excitation and detection scheme. The graphene film was transferred onto the endface of a ferrule with a 125-μm inner diameter. In contrast to the pre-tension induced in membrane that increased quality ( Due to zero pressure difference at both sides of the suspended membrane, the resonators with venting gas channels typically operate at lower pressures than a normal pressure, which also correspond to a comparatively low vibration damping condition. For instance, viscous damping is not the dominant dissipation process for the resonator reported in [Kn = λMFP/ldevice [λMFP is the mean free path of air molecules and ldevice is the graphene device characteristic length, ranges from 0.01 to 10. However, in reality, air damping is a significant viscous dissipation mechanism when nanomechanical resonators are operated in a moderate vacuum or near ambient conditions. Moreover, resonance characteristics in air micro-cavity also completely differ from those in a vacuum chamber [2 have found that the tension resulting from the fabrication process could increase the resonant frequency [These aforementioned experimental studies have significantly boosted the understanding of graphene resonance characteristics. However, in these experiments, graphene flakes are generally mechanically exfoliated by means of well-controlled forces ,11,14, a0−4 mbar . It shou0−4 mbar , whereinorted in  because /ldevice , where λ chamber ,25. Althrequency , especia2 ferrule endface with a diameter of 125 μm that is by far larger than several microns in previously reported resonators [2 mating sleeve was herein fabricated to make the comparative analysis of pre-stress-dependent resonance behaviors. Then, the resonance performance with varying pressure differences ranging from 0 to 199 kPa was tested by the presented simple optical fiber F-P interferometric resonance readout, which exhibited a pressure sensitivity of 1.18 × 105 kHz3/kPa that was by far superior to that represented by the cavity length changes for the same F-P sensor. The results validated the developed F-P resonator and the measured resonance frequencies in contrast to Q factors revealed the more strongly dependence upon the stress tensor in nanomembranes [Hence, in this paper, from the viewpoint of in situ applications of graphene-based F-P resonant sensors, we developed a simple and miniature micro-air-gap-based optical fiber F-P resonator covered with a chemical vapor-deposited (CVD) ~13-layer graphene circular diaphragm instead of beam-shaped diaphragm in  in ordersonators ,17,19,22embranes , in compembranes . 2 mating sleeve onto whose endface the graphene film, working as a light reflector, is adhered by van der Waals forces. The initial cavity lengths between the fiber end and ferrule endface for the two types of samples are confirmed by an optical spectrum analyzer (OSA)  via a 1-μm resolution translation stage. Then, the magnification scanning electron microscope  image of as-transferred multilayer film on the ceramic ferrule with a 125-μm diameter hole, as shown in ®). Note that the procedure of preparing the film from a commercial CVD 10–15-layer graphene sample  and transferring it onto the endface of a ferrule is same as that in [ that in . The thi that in . Ir in the F-P cavity in R1 and R2 are the reflectivities of the graphene membrane and fiber endface, respectively; Ii is the incident intensity; θ is the phase difference between two adjacent beams, and ξ is the coupling coefficient of cavity length loss [R2 is confirmed as 2.5% by measuring the interference of F-P cavity formed by two SMFs. As the lattice expands or contracts because of laser power-induced temperature change, the equilibrium positions of atoms and consequently the interatomic forces change, which induces the shift in the phonon energies and then membrane motion [t represents time; A, f, and ϕ are the vibration amplitude, frequency, and phase angle, respectively. According to the resonance motion of diaphragm, Ir in Equation (1) can be rewritten as:λ is the wavelength of incident light. In , Bunch egth loss . R2 is ce motion . Assuminδmem) of the suspended structure will modulate the reflectivity of the F-P interferometer created by both the graphene membrane and SMF. Since δmem creates a time-dependent variation of tension within the membrane, the δmem-related incident light intensity needs to be confined to an appropriate range rather than an undesirable higher light intensity, which otherwise will cause nonlinear mode coupling and internal resonances related with complex energy transfer between various vibrational modes [Q factor of the mechanical resonators are closely related with the dimension and physical properties of the resonator material, such as pre-tension, mass density and Young’s modulus, in addition to damping conditions. In this case, the resonance behaviors of pressurized graphene in air can be determined by extracting optically-actuated thermally-induced membrane displacements perpendicular to the graphene diaphragm.From Equation (2), the resulting motion  and 10−2 Pa (vacuum pressure), where viscous damping was remarkably different since the Knudsen number Kn was confirmed as around 0.0006 and 6350, respectively. For the former, viscous damping becomes significant. On the contrary, the latter is not in the viscous damping regime. Then regarding the limited impact of air damping on resonance behaviors, the conventional F-P sensor probe was also utilized to further investigate the pressure-sensitive resonance behaviors at pressures ranging from 101 to 300 kPa, i.e., a relative pressure range of 0–199 kPa, with a corresponding lower Kn value ranging from 0.0002 to 0.0006.f and Q of the two clamped circular graphene diaphragms at two typical pressures (105 and 10−2 Pa) representing viscous and free molecule flow damping regimes because of Kn = 0.0006 and 6350, respectively. Note that the two diaphragms are correspondingly adhered to the ferrule endfaces in the two types of sealed and open F-P probes as mentioned in The photothermal displacement spectrum from the graphene resonator contributes to analyzing the dependence on the primary effect suffering from the membrane thickness-dependent bending rigidity or initial pre-tension. Q factor of graphene-based nanomechanical resonators spans from 2 to 2 × 105, as it is strongly dependent on the sample fabrication and measurement conditions [Q (around 3.2–3.4) when the air pressure is reduced to 10−2 Pa from an atmospheric pressure, thereby implying a lower pre-stress value which is closely concerned with the interfacial energy between graphene and supporting substrates. In accordance with the membrane-to-plate behavior of 2D mechanical resonators with certain thickness [fair, fmem and fplate are the resonance frequency in air damping, XXX, and XXX, respectively; ρ is the mass density of graphene with certain adsorbates; ρair is the density of air; r, S, E, t, and υ are the radius, tension, Young’s modulus (1 TPa), thickness and Poisson’s ratio (~0.17) of graphene, respectively; Γ is a non-dimensionalized added virtual mass incremental factor as a function of mode shapes and boundary conditions. Note that Γ = 0.746 in the fundamental resonant mode f0,1. Hence, the resonance frequencies at various pressures enable the estimation of ρ and S simultaneously by fitting measured frequencies, along with the established continuum model. In this way, we estimated the pre-tension S0 to be 1.32 × 10−5 N/m for the resonator mentioned in ρ = 10ρg, where ρg is the mass density of pure graphene. The resulting equivalent mass density is similar to graphene resonators in [For the F-P resonator with open cavity configuration, no pressure gradient exists within the membrane, which indicates that the mechanical response in nditions . Herein,hickness , the reshickness :(3)fairators in ,12,13, wQ factor of ~18.5 with a higher pre-tension S0 of 0.022 N/m at normal pressure on basis of the aforementioned model. Furthermore, the Q factor increases to ~75.37 by lowering the atmospheric pressure to 10−2 Pa, although the value is much less than those reported in previous literature [−5 Torr [f under tensile tension, which conforms to the trend obtained in Equation (3). The small frequency shift of about 15 kHz also partly results from the limited pre-tension. As a result, the benefit of reduced air damping with decreasing pressure will gradually become remarkable, particularly for the resonator with greater pre-tension. Note that the tension is primarily caused by the fabrication process, where the friction between the graphene and the oxide surface stretches the graphene sheets across the trench or hole [2 ferrule-supporting suspended graphene was polished with diamond sheets in different grit sizes of 3 and 1 µm . The residuals on polished surface were cleaned by ultrasound equipment. ®, MS60, Guangzhou, China) of a ferrule endface before and after polishing. Small burrs and impurities at the edge of circular hole can be clearly found in In contrast, the F-P resonator with sealed cavity configuration in terature . A likel[−5 Torr ,13,17,19[−5 Torr ,16,38. IR-square of about 92.7%. Additionally, in terms of small fluctuations relative to the fitted output, a decent linearity of 5.15% was obtained due to fast dynamic response of the suspended membrane. After that, a conventional F-P interferometric readout scheme was also performed for the fabricated sensor to evaluate the presented F-P resonant detection. Referring to R-square of 53.5% in the negative going direction. Unfortunately, the sensor demonstrated a more inferior response in the positive going direction. It can be clearly observed that the measured three time results deviated from each other, particularly at the second cycle where an almost zero response occurred. For the remaining two cycles of positive measurements, an extremely low sensitivity of around 1.57 nm/kPa was recorded. It should be pointed out that during the F-P interferometric experiment, a broadband laser source  was used to illustrate the sensor, and the reflection spectrum was then monitored by an OSA (AQ6370C) with a wavelength resolution of 0.02 nm. With regard to the clamped circular graphene film made of a linear isotropic elastic material, based on the spherical shell equation, the relationship between the diaphragm deflection ω and the pressure change P is defined by [σ0 is the pre-stress of graphene that was confirmed as ~0.24 GPa on basis of the measured resonance frequency. Then a theoretical pressure sensitivity of 1121 nm/kPa was solved according to Equation (4), which exceeded, by far, the aforementioned measured 1.91 or 1.57 nm/kPa. The resulting load-deflection behavior primarily resulted from a lower pressure difference that was concerned with the slow air leakage of F-P cavity covered with large-sized CVD multilayer graphene film. Moreover, the phenomenon became more serious in pressure rise measurement than in pressure drop measurement because of longer air balance process in the air chamber. Although recent advances in graphene synthesis and processing have enabled demonstration of atomically-thin 2D membranes showing mechanical sturdiness and hermetic sealing [2 in cross-sectional area is generally chosen to reduce the possible risk of leakage from random defects in large-area nanomembranes [Referring to fined by :(4)P=4σ sealing ,41, a liembranes  or poorlf and Q) over the tested pressure range. Owing to the rising pre-stress tuned by the polished substrate, the measured f was increased to more than 500 kHz well above the frequency of 60–80 kHz in ρ = 10ρg mentioned above, an improved pre-tension S0 for the F-P resonator using the polished surface of a ferrule as film substrate was calculated to be 1.05 N/m. However, a slight frequency deviation up to about 33 kHz existed between them, which is to a great extent due to the presence of non-uniformly distributed adsorbates or polymethylmethacrylate (PMMA) residuals and the stress distributions in the graphene-substrate interface during membrane transfer and sensor fabrication as mentioned above. In the light of the aforementioned Equation (3) relating to fmem, fplate and f in the membrane, plate, and cross-over regime, respectively, it can be inferred from 5 kHz3/kPa with a good linearity error of 5.16% was achieved in the tested range, which is far below the theoretical predicted sensitivity of 1.32 × 107 kHz3/kPa in terms of the Equation (5) as follows:E and ρ are assumed as 1 TPa [ρg as mentioned above, respectively. The great deviation between the measured and predicted resonant frequencies possibly results from the equivalent density-dependent impurity and defect effects in transferred suspended graphene with a large membrane dimension, the pre-tensioned-membrane size effect and boundary instability of clamped circular graphene diaphragm in air damping. It should be added that the discrepancy between the measured values and predicted ones is also dependent on the presence of higher eigenmodes featuring the nonlinear oscillation mode coupling with rolling frequency multiplication components, which results from the alternating tension in graphene because of higher vibration amplitude comparable to the thin membrane thickness [hickness ,44. Accohickness , therebyQ factor can be determined by the full width half-maximum of the Lorentzian resonance peak; however, there is no exact dependence of Q on resonance frequency. However, as illustrated in the inset in Q factor decreases as a function of external pressure that is represented by pext, approximately proportional to 1/pext, which is well in agreement with the variation in [Q factor from ~13.3 to ~16.6 fluctuates around 14.9, which is weakly associated with fundamental frequencies. The average Q factor is ~14.9 for the resonator surrounding the air at normal temperature, which is obviously better than that with extremely low pre-tension (1.32 × 10−5 N/m) in Q factors are also superior to the results at atmospheric pressure in [Q factors from 17 to 600 and resonant frequencies from 7 MHz to 206 MHz at room temperature [Q factor for the resonator presented in this paper is relatively significant in the limited test frequency range. Note that it is known that the increase in membrane size enables to reduce f and Q in viscous damping, but to enhance Q in free molecule flow damping. Consequently, engineering structural size-dependent tensile stress in membrane-type resonators can further elevate Q by raising instinct energy stored in graphene membranes [Then, according to the inset in ation in . Furtherssure in ,22. Thenperature , the chaembranes .Q factor values are by far less than most of the results reported in previous literatures. The resulting primary reason is that those resonators in [Kn > 104. However, according to the remarkably reduced Q factors in reported experimental results, the resonators in [Kn decreases to the range of 10–50. Then when Kn < 0.01, air damping becomes a significant viscous dissipation mechanism, which causes the Q factor to drop sharply. Thus, the presented resonator in this paper exhibited a same order of Q factor as those working at normal pressure in [Q factor. This suggests that the adhesion conditions at the graphene-substrate interface should be controlled reasonably. Unfortunately, since the adsorbates would generate additional masses and tensions that will result in the dispersion of resonant responses among different samples, the damping mechanism-dependent Kn should be designed optimally by engineering membranes suspended on F-P resonators. In consideration of the film integration in resonators, the facile-fabricated and in situ F-P resonator is vital for dynamical performance evaluation in actual applications, along with its corresponding simple F-P photothermal actuation and detection scheme. Compared with previous graphene nanomechanical resonators reported in It is worth pointing out that the experimental results elucidate that a simple and miniature open or sealed optical fiber F-P pressure-sensitive resonator with large-scale graphene or other 2D diaphragms could be successfully developed without need of complicated free-space optical actuation experimental setups. In order to further elaborate the uniqueness of the developed F-P resonator, some typical experimental works on graphene nanomechancial resonators were summarized and listed in ators in ,11,12,15ators in ,22,31 shssure in ,22,31 anssure in . The thi−2 Pa due to the free molecule flow damping. Then according to the interaction with the air, the pressure-induced modal frequencies from 509 to 542 kHz were further identified with little dependence on Q ranging 13.3–16.6. Despite lower measured Q factors in comparison to the value up to 2 × 105 reported in previous studies where vacuum pressure of <10−6 Torr or low temperature of <50 mK were generally set, the experimental results herein demonstrated a pressure sensitivity of 1.18 × 105 kHz3/kPa with a linearity error of 5.16%. Although its sensitivity was far below the predicted one possibly because of impurity and defect effects in graphene, pre-tensioned-membrane size effect and boundary instability of clamped diaphragm in air damping, the fabricated graphene-based F-P resonator demonstrated a preferable response to applied static pressures, compared with a conventional F-P interferometer with same micro air-cavity configuration. Therefore, this study could shed light on the resonance behavior in situ observed in 2D nanomechanical resonators in air damping, along with strain-tunable substrate microstructure and thin film vacuum encapsulation.Two types of simple, miniature, and in situ mechanical F-P resonators with open and sealed cavities were fabricated to characterize the resonance behaviors of a nanothick graphene diaphragm suspended onto a large-area 125-μm inner diameter ferrule endface by using simple optical interference excitation and detection. The effects of gas pressure-induced damping and pre-tension in membranes on resonance characteristics were experimentally investigated in the absolute pressure range of 101 to 300 kPa at room temperature. Due to a smaller pre-tension in membrane, the micro air-cavity-based graphene device exhibited the lower resonance frequency of 59 kHz but remarkable Q factor of ~75.37 at 10


Long interspersed nuclear elements-1 (L1s) are a large family of retrotransposons. Retrotransposons are repetitive sequences that are capable of autonomous mobility via a copy-and-paste mechanism. In most copy events, only the L1 sequence is inserted, however, they can also mobilize the flanking non-repetitive region by a process known as 3′ transduction. L1 insertions can contribute to genome plasticity and cause potentially tumorigenic genomic instability. However, detecting the activity of a particular source L1 and identifying new insertions stemming from it is a challenging task with current methodological approaches. We developed a long-distance inverse PCR (LDI-PCR) based approach to monitor the mobility of active L1 elements based on their 3′ transduction activity. LDI-PCR requires no prior knowledge of the insertion target region. By applying LDI-PCR in conjunction with Nanopore sequencing (Oxford Nanopore Technologies) on one L1 reported to be particularly active in human cancer genomes, we detected 14 out of 15 3′ transductions previously identified by whole genome sequencing in two different colorectal tumour samples. In addition we discovered 25 novel highly subclonal insertions. Furthermore, the long sequencing reads produced by LDI-PCR/Nanopore sequencing enabled the identification of both the 5′ and 3′ junctions and revealed detailed insertion sequence information. Nevertheless, somatic L1 insertions can drive tumorigenesis and high L1 expression is emerging as a common trait of several cancers2. For an L1 to be potentially active, it needs to be a full-length element with a 5′ promoter and two intact open reading frames, ORF1 and ORF2, terminating with a 3′ polyadenylation signal.Long interspersed nuclear elements (LINE)-1, also known as L1 elements, are active mobile repeat elements in the human genome. Germline L1 polymorphic insertions are suggestive of L1’s contribution to genomic diversity4. Upon activation, the L1 is transcribed into an RNA intermediate, which is then translated into two proteins, ORF1p and ORF2p. The L1 mRNA, ORF1p and ORF2p form a ribonucleoprotein complex that nicks the DNA at the target location via the endonuclease function of ORF2p. The polyadenylated 3′ end of the L1 mRNA anneals to the T-rich region of the target site, which then acts as a primer for reverse transcription5. Thus, this process, referred to as target-primed reverse transcription (TPRT), integrates L1 sequence into the target region with a signature polyA sequence marking initiation of reverse transcription. TPRT sometimes is accompanied by a process called twin-priming that leads to inversion of the L1 inserts. This results when one overhang produced on the target region by ORF2p-mediated staggered double-strand cleavage serves as a T-rich or polyT primer for TPRT, and the other anneals to the internal L1 mRNA sequence serving as an internal primer6.Out of more than 500,000 copies of L1 sequences in the human reference genome only 90 to 100 are potentially active3. These hot L1 elements are of considerable interest as they show frequent somatic insertions in various types of cancers  , also shown to be an active element in an in vitro retrotransposition assay3, is highly active in colorectal cancer11. It was possible to identify this particular L1 (hereafter referred to as TTC28 L1) as the source of the insertions by using short paired-end read sequencing based on the mobilization of its non-repetitive (unique) 3′ flanking sequence, via a mechanism known as 3′ transduction14. 3′ transduction occurs when the canonical 3′ polyadenylation signal of the source L1 is weak, causing the transcription machinery to skip it and to continue transcribing the non-repetitive region downstream in the 3′ flanking region until it reaches a stronger polyadenylation/termination signal. Consequently, some of this unique sequence is included in the RNA intermediate and subsequently incorporated into the new chromosomal location, thereby serving as a unique sequence tag that reveals the L1’s origin.Not all potentially active L1 elements contribute equally to retrotransposition events however, and only few “hot” L1 elements are responsible for most retrotranspositionsers Fig. 7–11. Par15, L1-seq.16, RC-seq.17, transduction-specific ATLAS18, TIP-seq.19) and used to identify somatic L1 insertions, but they are quite extensive if the aim is to simply assess the activity of a few L1 loci. Furthermore, all current methods targeting L1 insertions are limited in their capacity to simultaneously resolve full insertion sequence. Thus we have developed a direct molecular approach to detect 3′ transductions from specific L1s and hence monitor their activity; this method requires no prior knowledge of the insertion target regions. We apply long-distance inverse (LDI)-PCR20 to a particular source L1 (TTC28 specific L1 in this study) by targeting inverse primers to its frequently transduced 3′ flanking sequence (here referred to as the “unique tag”)  can identify 3′ transductions, detecting these L1-mediated insertions is still a great challenge, principally due to the repetitive nature of L1 sequences and our limited capacity to sequence long fragments of DNA. Several next-generation sequencing strategies targeting young L1s or 3′ transduced regions have been developed (ATLASg”) Fig. . Note, h21 to select two colorectal tumour samples with high number of TTC28 L1 3′ transductions (hereafter referred simply as insertions) for our proof-of-concept analysis and comparison. By selective amplification of the transduced region using LDI-PCR  and three different primer pairs (Supplementary Table TTC28 L1 (the “native PCR product”) was observed in almost all the samples digested with PstI and SacI . After filtering the LDI-PCR.py calls, we were able to identify 14 out of 15 previously detected insertions. Additionally, we detected 25 novel insertions not identified by WGS, despite visual inspection of the paired-end read data   . Subsequently we performed allele-specific PCR and Sanger sequencing on another ten insertions, of which we successfully sequenced the insertion-to-target junctions for six of them Table . Again, TTC28 L1 3′ transductions used either the 6th or the 9th polyadenylation signal instead of the canonical polyadenylation signal  TPRT with twin-priming, exemplified by the L1 insertion at CNTNAP2 locus in chromosome 7  TPRT, exemplified by the L1 insertion at RNA Fig.  and wereRNA Fig. . In addince Fig. . Upon clnce Fig. . This twion Fig. . We alsoion Fig. . MicrohoIn order to interpret the advantages and disadvantages of long read sequencing, we compared the local assembly of paired-end read data to the LDI-PCR/Nanopore consensus sequences of those insertions in tumour sample c985T that were detected by both methods. After local assembly, we were able to reconstruct four out of seven insertions into one contiguous fragment (contig) (Supplementary Table 10 or (b) when it involves 3′ transduction of the unique flanking sequence11. Thus, in order to determine the activity of a particular source L1, one needs to scan the genome of interest for either of the aforementioned signs. Assessing the activity of a particular source L1, in particular when active in a tumour type, can be of great interest, as these L1 insertions can cause tumorigenic events or serve as a clinical biomarker24. However, the specific detection of L1 3′ transductions and/or sequencing the whole length of L1 insertions remains a great challenge. In this study we used a LDI-PCR based assay to study the activity of a source L1 at the TTC28 locus, previously shown to be highly active in colorectal cancer, and tested it on two colorectal cancer samples with already published whole genome sequencing data21.Due to the repetitive nature of L1 elements and their abundance in the human genome, it is possible to determine the lineage or source of the L1 insertions either (a) when the insertion contains signature single nucleotide polymorphisms associated with the source L1TTC28 L1 mediated 3′ transductions, and also discovered 25 3′ transductions not detected by WGS (Table TTC28 L1 3′ transduction is due to (a) the nature of nucleotide sequence downstream of the L1 3′ end, possibly due to many small stretches of Ts present which could complement with a stretch of As generated by the ORF2p endonuclease action on the target region which in turn can be used as the second or “internal” primer leading to twin-priming, or (b) detection of more L1 insertions with these inversion properties than by conventional methods due to the sequencing of the whole inserted sequence. We were also able to sequence the entire source TTC28 L1  polyA present in the reference genome.SacI) and two primer pairs on one sample (c985T), we were able to detect only 4 insertions out of 8 detected by WGS analysis. Detection sensitivity was substantially improved by updated sequencing chemistry, the use of two additional restriction enzymes and by designing more primers that covered additional polyadenylation signals in a well-dispersed manner.LDI-PCR/Nanopore success principally relies on (i) a careful selection of the restriction enzymes so as to produce PCR-amplifiable target templates and (ii) the design of multiple primer pairs covering several downstream polyadenylation signals predicted by tools such as polyadq. In our initial pilot LDI-PCR/Nanopore sequencing experiment using one restriction enzyme To conclude, we demonstrated that LDI-PCR/ Nanopore sequencing is suitable for sequencing the entire L1 insertion and for detecting highly subclonal events. Consequently, applying LDI-PCR in conjunction with Nanopore sequencing in larger sample sets and different tumour types enables a more detailed characterization of L1 insertions providing new insights into L1 biology and cancer genetics.26. The tumours were fresh frozen and the corresponding normal tissues were obtained from blood (c985T) and from colon tissue (c368T). The study was reviewed and approved by the Ethics committee of the Hospital district of Helsinki and Uusimaa, Finland. A signed informed consent or authorization from the National Supervisory Authority for Welfare and Health was obtained for all the samples.The colorectal adenocarcinoma (CRC) samples utilized in this study were obtained from a population based series of 1042 CRCs previously describedTTC28 L1, genomic DNA was separately digested by three restriction enzymes: SacI, PstI and NsiI. SacI and PstI make a 5′ cut at ORF1 of the L1 and a 3′ cut downstream of the unique tag producing a native restriction fragment of 5.7 kb and 6.3 kb respectively, whereas NsiI makes a 5′ cut 3.1 kb upstream of the intact L1 sequence and a 3′ cut downstream of the unique tag generating a native restriction fragment of ~10.2 kb . Primers were designed using Primer3 (http://primer3.ut.ee) and their specificity was checked using NCBI Primer-BLAST (available at http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Since there were several polyadenylation signals at the 3′ flanking region of the L1  were pooled into nine different barcodes in equal molarity. Tape Station 2200 (Agilent Technologies) was used to estimate the relative molarity based on the fragment distribution in each reaction. Libraries were constructed according to the manufacturer’s instructions using SQK-LSK108 and EXP-NBD103 sequencing and barcoding kits (Oxford Nanopore Technologies). Equal molarity was preserved throughout the protocol. The MinION Flow cell (FLO-MIN106) was run for 6 hours using MinION Mk1B. The raw signal from MinION was basecalled with ONT Albacore Sequencing Pipeline Software (version 1.0.2). The reads passing base calling were aligned to GRCh37 genome reference augmented with viral and 1000 genomes decoy sequences. The alignment was performed with bwa mem v0.7.12 using option -x ont2dTTC28 L1 (22:29060420–29067335) and at least one alignment to a single target locus in the genome. The read can have multiple supplementary alignments to the target locus but only if located in close proximity of each other . All considered alignments had to have mapping quality of at least 20. The insertion breakpoint was defined by the location where the alignment switched from TTC28 L1 to the target locus and vice versa for each hallmark read. All insertion breakpoints were clustered together allowing a maximum gap of 3 kb from all reads defining the different insertion locations. The most frequent genomic coordinate was called as the insertion breakpoint defining each LDI-PCR insertion call. The software is available at https://github.com/kpalin/LDI-PCR-call and https://github.com/kpalin/ampcorrect.We separated the reads produced by different samples, PCR primers and restriction enzymes by using the sequencing barcodes and comparing the read mapping with restriction enzyme cut sites in the reference genome. To systematically detect the insertions, we developed the LDI-PCR.py software which identifies reads that display hallmark features of LDI-PCR products Fig. . Hallmar28, to obtain accurate consensus sequences for the amplicons. Briefly, ampCorrect uses sumaclust29 (http://metabarcoding.org/sumatra) to cluster the reads, requiring 60% sequence similarity and poaV2 -do global30 to align multiple reads. The consensus sequence is treated as corrected sequence of the analysed amplicon. The processed sequences were aligned to the human reference genome using bwa mem with default parameters. The analysis of the insertion characteristics was performed on a consensus sequence which was constructed from 20 random candidate insertion call reads using UCSC BLAT (https://genome.ucsc.edu/cgi-bin/hgBlat) , several insertion calls were present in both samples. In order to circumvent this issue, in cases where the insertion call was present in two samples, only the calls coming from the sample that contained at least 95% of the reads were included in further analyses. Insertions in mitochondria and unplaced sequence were filtered away. All insertions that fulfilled the abovementioned criteria in at least one reaction were defined as candidate insertions and selected further for consensus sequence analysis of the insertion characteristics. The reads from the candidate insertions were processed with ampCorrect, a Nanopore read correction method similar to nanocorrect31, we analysed how many 3′ coordinates of the L1 insertions were located within a 10–30 bp window downstream of each of the polyadenylation signal  located downstream of the L1 sequence  the reaction with higher read count was used.et al.21 to select for LDI-PCR and Nanopore sequencing those colorectal cancer samples with a high number of somatically acquired insertions originating from TTC28. Structural variant breakpoints located at the 3′ end of the TTC28 L1 were extracted to calculate the number of transductions. The 3′ end of the L1 was defined as GRCh37 coordinates 22:29065455–29066124. To examine whether the novel candidate insertions were detected by WGS or not, we performed a thorough visual inspection of the paired-end read data using BasePlayer32.We utilized the WGS dataset described in Katainen 33. All hash lengths within default parameters  were tested and the hash length that produced the longest and most contiguous contig was selected for each insertion33 (Supplementary Table https://genome.ucsc.edu/cgi-bin/hgBlat) to the GRCh37 genome reference.In order to compare LDI-PCR Nanopore sequencing results to WGS data we performed local assembly of the WGS data. We selected those chimeric reads and discordant read pairs that aligned within 1kb upstream and downstream the predicted insertion breakpoint, with the exception of one insertion (chr15:97602708) where, due to a long target site deletion, a 3kb window was used. The local assembly of the reads was performed using Velvet 1.2.10http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). Primer sequences are in Supplementary Table http://www.freewarefiles.com/FinchTV_program_17782.html).Two approaches were used to validate novel candidate insertions detected by LDI-PCR but not by WGS: (i) First, primer pairs were designed on the target genomic region across the insertion breakpoint and sequenced by Nanopore. The library was prepared as described in the section “Oxford Nanopore MinION™ sequencing”. This approach included all candidate subclonal insertions, however only 3/25 novel candidate insertions were validated (ii) Second, primers were designed based on the consensus sequence, with one primer at the target site and the other primer at the inserted sequence, followed by Sanger sequencing of the resulting PCR product; this was performed for 10 selected novel candidate insertions. Primer pairs were designed with primer3Plus (The datasets generated during and analysed during the current study are available from the corresponding author on request.Supplementary InformationSupplementary dataset


Programmed death-ligand 1 (PD-L1) is thought to play a critical role in immune escape by cancer, but whether PD-L1 expression can influence prognosis of patients with solid tumors is controversial. Therefore, we meta-analyzed available data on whether PD-L1 expression correlates with overall survival (OS) in such patients. PubMed, EMBASE and other databases were systematically searched for cohort or case-control studies examining the possible correlation between PD-L1 expression and OS of patients with solid tumors. OS was compared between patients positive or negative for PD-L1 expression using scatter plots, and subgroup analyses were performed based on tumor type and patient characteristics. Data from 59 studies involving 20,004 patients with solid tumors were meta-analyzed. The median percentage of tumors positive for PD-L1 was 30.1%. OS was significantly lower in PD-L1-positive patients than in PD-L1-negative patients at 1 year (P = 0.039), 3 years (P < 0.001) and 5 years (P < 0.001). The risk ratios of OS  were 2.02 (1.56-2.60) at 1 year, 1.57 (1.34-1.83) at 3 years and 1.43 (1.24-1.64) at 5 years. Similar results were obtained in subgroup analyses based on patient ethnicity or tumor type. The available evidence suggests that PD-L1 expression negatively affects the prognosis of patients with solid tumors. PD-L1 might serve as an efficient prognostic indicator in solid tumor and may represent the important new therapeutic target. Immune co-stimulatory and co-inhibitory receptors determined the functional outcome of T cell receptor (TCR) signaling and immune surveillance . Tumors PD-L1 also plays a key role in binding to PD-1 receptors expressed on activated T cells in T cell co-suppression and depletion –11. PD-LIn addition to serving as a therapeutic target, PD-L1 may also be useful as a prognostic biomarker . HoweverA total of 1,258 records were retrieved from PUBMED, EMBASE, Web of Science and EBSCO , 3 years (P < 0.001) and 5 years  were PD-L1-positive and 3,206 (64.4%) were PD-L1-negative. PD-L1 expression was associated with significantly worse OS at 1 year (P = 0.004), 3 years (P = 0.005), and 5 years  were PD-L1-positive and 1,404 (59.3%) were PD-L1-negative. PD-L1 expression was associated with significantly worse OS at 1 year (P = 0.017), 3 years (P = 0.010) and 5 years , 3 years P = 0.005) and 5 years , 3 years (P = 0.040) and 5 years  primary solid tumors in human patients; (2) The main content of the articles is to analyze the relationship between the expression of PD-L1 and the prognosis of solid tumors in patients; (3) a hospital-based or population-based case-control or cohort design, regardless of sample size; (4) immunohistochemical assay of PD-L1 expression as high and low PD-L1 expression; (5) all patients underwent surgery; and (6) adequate reporting of overall survival (OS) data. When eligible studies involved overlapping patient populations, only the most recent or complete report was included. Studies were excluded if they were letter, summary of meeting and review; if they were published in a language other than English; or if they failed to report adequate data; or they investigated metastatic tumors. Gray literature  was not included into this study. Reference lists within identified articles were also searched manually to identify additional articles.The primary outcome in the meta-analysis was OS. This outcome was compared between patients showing high or positive PD-L1 expression and patients showing low or no expression, as defined within the individual studies.Two researchers  independently screened studies for inclusion. Disagreements were resolved by discussion and, when necessary, consultation with a third author (S.Z). The first author's name, year of publication, country, number of patients, and tumor type were extracted from each study, and OS results for 1, 3 and 5 years were extracted from tables or Kaplan-Meier curves.2statistics. Survival results were analyzed using scatter plots generated in Prism 5 . The results for different patient groups were compared using the log-rank test. The threshold of statistical significance was defined as P < 0.05.Forest plots of OS were generated using RevMan 5.3 . Weighted risk ratio (RR) estimates were generated from pooled data using Mantel-Haenszel random-effects meta-analysis, unless no statistically heterogeneity, in which case fixed-effects meta-analysis was performed. Statistical heterogeneity in meta-analyses was assessed using Cochrane's Q and I


Biologists have taken the concept of organism largely for granted. However, advances in the study of chimerism, symbiosis, bacterial‐eukaryote associations, and microbial behavior have prompted a redefinition of organisms as biological entities exhibiting low conflict and high cooperation among their parts. This expanded view identifies organisms in evolutionary time. However, the ecological processes, mechanisms, and traits that drive the formation of organisms remain poorly understood. Recognizing that organismality can be context dependent, we advocate elucidating the ecological contexts under which entities do or do not act as organisms. Here we develop a “contextual organismality” framework and provide examples of entities, such as honey bee colonies, tumors, and bacterial swarms, that can act as organisms under specific life history, resource, or other ecological circumstances. We suggest that context dependence may be a stepping stone to the development of increased organismal unification, as the most integrated biological entities generally show little context dependence. Recognizing that organismality is contextual can identify common patterns and testable hypotheses across different entities. The contextual organismality framework can illuminate timeless as well as pressing issues in biology, including topics as disparate as cancer emergence, genomic conflict, evolution of symbiosis, and the role of the microbiota in impacting host phenotype. Biologists have taken the definition of organism for granted for most of the history of the study of life. However, at a time when biologists have discovered that bacterial‐eukaryote symbiosis is near‐universal, that cancer cells may collaborate to create tumors, and that microbes can communicate and perform collective actions, the concept suddenly seems less clear. Did my genes stuck in my mother affect my brother's development  form a new entity characterized by high cooperation and low conflict among the parts .We argue that a given assemblage of parts will or will not behave as an organism‐like unit, depending upon the specific ecological conditions in time and space in which it occurs. Contextual organismality focuses on studying the traits and mechanisms that are associated with such transitions. For instance, single‐celled microbes in groups, widely regarded as competitors, can show highly cooperative interactions under conditions of starvation, leading to fruiting bodies that allow dispersal and survival of a subset of cells Kaiser . When anDoes the group's cooperation‐conflict level change?When and under what ecological context does it change?What were the important traits that enabled that change?Do different biological groups change under similar circumstances, or are similar traits important?The relevant contexts that determine a group's cooperation‐conflict dynamic vary according to its composition, but can include life stage (development), resource availability, population size, and interactions with other species. Examining different types of biological entities in different contexts reveals exciting new questions that emerge by viewing the phenomenon through the contextual organismality lens:Contextual Organismality in Practice). The answers to these questions can shed light on some of the major challenges in the study of organismality and also generate novel, testable hypotheses to gain further insights into interactions among biological entities.These questions form the basis of investigations guided by the conceptual organismality framework, as we will show in a subsequent section by applying them to biological examples , and the hawkmoth Manduca sexta shifts over developmental time with regard to the relative importance of cooperation versus conflict  due to pollen transport between flowers  in many forms .Thus, a lack of context dependency can be used as an indicator of an organism, that is, a group that preserves high cooperation and low conflict among the parts across widely divergent contexts. This conceptualization of the organism reconciles the fluidity of contextual organismality with organismality approaches that employ fixed definitions of an organism . Potential model systems to test this prediction are interspecific mutualisms that vary in their degree of unification  for the similar responses of cells to starvation across the tree of life.What is an organism?” As with many other fundamental questions, it is unlikely that a single answer will be forthcoming. However, tackling this question has great potential to generate knowledge. An analogy with the question “What is a species?” is appropriate . After over three centuries of study, biology is arguably further away from an answer. Now there are multiple definitions, called species concepts. What has been gained is a breadth and depth in the knowledge of the process of speciation and all the different ways it can happen (De Queiroz It is our hope that the contextual organismality framework will contribute to expanding empirical and synthetic research into one of the fundamental questions of biology: “When is an organism?” to the research agenda. The answer lies in the mechanisms of organismality, which can inform issues as disparate as cancer emergence, the role of the microbiome, and genomic conflict, addressing both pressing and timeless questions in the study of life.Likewise, it is our hope that contextual organismality serves as a useful framework for understanding the process by which organisms emerge. We advocate adding the important question “Associate Editor: R. AzevedoHandling Editor: R. Shaw


Purpose. To assess the effect of three-dimensional (3D) printed personalized moisture chamber spectacles (PMCS) on the periocular humidity. Methods. Facial computed tomography (CT) scanning was conducted on 10 normal subjects. PMCS was designed based on volume rendered CT images and produced using a 3D printer. Periocular humidity of PMCS and commercially available uniformed moisture chamber spectacles (UMCS) were measured for 30 minutes via microhydrometer. Results. The mean ambient humidity was 15.76 ± 1.18%. The mean periocular humidity was 52.14 ± 3.00% in PMCS and 37.67 ± 8.97% in UMCS. The difference was significant (P < 0.001). Additionally, PMCS always demonstrated lower humidity than dew points. Conclusion. PMCS made by 3D printer provides appropriate fitness for the semiclosed humid chamber. PMCS showed higher performance than UMCS. The wearing of PMCS would be an effective method to provide high enough periocular humidity in low humidity environment.  Dry eye is a common ocular disease prompting millions of individuals to seek ophthalmological care . ArtificKorb et al. reported that increased periocular humidity using modified swim goggles resulted in an increase in tear film lipid layer thickness and moderate to total relief of dry eye symptoms . TsubotaUnlike general spectacles, moisture chamber spectacles need to be personalized in shape and size of frame for best performance. The tear retention effectiveness of moisture chamber spectacles is determined by the extent of proper semiclosed chamber formation. The formation of an appropriate chamber depends on how well the spectacles fit the facial contour. There are several commercially available moisture chamber spectacles; however they are single size or minimally adjustable. Previous studies were also performed using spectacles with single or several size variations , 3.Three-dimensional (3D) printing was invented in the 1980s and has shown marked growth in recent years. 3D printing has been advancing in customized product printing and has proved to be effective for medical applications –6. RecenIn this study, personalized moisture chamber spectacles (PMCS) were produced using facial computed tomography (CT) scanning and 3D printing technology. The effect of PMCS on periocular humidity was evaluated and the clinical implication was discussed.This study was approved by the Institutional Review Board of research ethical committees of Aeromedical Center, Republic of Korea Air Force, and conformed to the tenets of the Declaration of Helsinki (ASMC-15-IRB-001). Ten normal subjects who have no history of evidence of ophthalmic disease were recruited.Facial CT scanning for PMCS was conducted on the participants. Facial CT scanning was performed using a 64-channel CT scanner . After conventional CT scan, 3D reconstruction was processed and facial contour was obtained using volume rendering tool, which was incorporated in the workstation .2 and held 0.85 g of distilled water. Silicone part of moisture chamber was constructed on CAD and the mold was printed. Then silicone  was infused into the mold and silicone chamber was formed.The spectacles were designed using computer-aided design (CAD) program . PMCS consisted of mainly 4 parts, that is, frame, side panel, moistened sponge block, and silicone chamber . All parThe present study was designed as cross-over test. The subjects wore a sequence of different moisture chamber spectacles. Commercially available uniformed moisture chamber spectacles (UMCS)  and PMCS were worn naturally for 30 minutes while periocular humidity and temperature were measured. UMCS were worn once again with gentle pressure on the spectacles frame in order to measure periocular humidity with nearly perfect sealing of moisture chamber. The order of the wearing spectacles was randomized. There was an interval of 30 minutes, prior to wearing other spectacles. The microhydrothermometer  was attached to the backside of the spectacles lens . Left eytd = dew point, t = temperature, and RH = relative humidity): t” and ambient temperature for “td.” The calculated RH and measured periocular humidity were compared.The excessively high periocular humidity induces condensation on the spectacles lens and visual blur. The dew point is the temperature at which dew forms. A simple equation for the relationship between relative humidity and the dew point is as follows   and Bonferroni post hoc test were performed for comparisons among measurements. Shapiro-Wilk was used for normality test. Statistical analysis was conducted, using SPSS Statistics Version 21 . All tests were two-tailed and The mean age of participants was ±years. Eight subjects were males and 2 were females. Facial characteristics including interpupillary distance, bitemporal distance, and temple length were described in P < 0.001), (The hydrometer had an accuracy of ±3% RH and a repeatability of ±1% RH. Measurements of periocular humidity over time were shown in  0.001), .P = 0.002). However, the difference of periocular temperature between pressurized wearing of UMCS and natural wearing of PMCS was not significant (P = 0.071), (The thermometer had an accuracy of ±0.5°C and a repeatability of ±0.3°C. Measurements of periocular temperature over time were shown in  0.071), .Measured periocular humidity and calculated humidity level that starts to cause the condensation on the spectacles were shown in The wearing of moisture chamber spectacles can be helpful to improve ocular discomfort associated with dry eye. Based on the definition from the dry eye workshop (DEWS), dry eye disease is defined as multifactorial disease that is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface . The DEWIn this study, PMCS increased absolute value of 36.39% periocular humidity, as compared to the ambient humidity. Moisture chamber spectacles may be particularly helpful to reduce artificial tear use for severe dry eye, involving Sjögren's syndrome or lagophthalmos patients, and ultra-low humidity environmental worker including laboratory employee  or aircrAppropriate fit is the most critical aspect in moisture chamber spectacles. The loose fitness of spectacles reduces relative humidity in the moisture chamber. In the present study, PMCS showed significantly higher periocular humidity than UMCS, as predicted. Additionally, UMCS demonstrated wide individual differences of periocular humidity (standard deviation = 8.96), while PMCS showed narrow individual variations (standard deviation = 3.00). In contrast, tight fitness of spectacles induces excessively high relative humidity, resulting in condensation as well as discomfort. Moisture chamber should be a semiclosed, and not a completely closed space due to condensation. In this study, pressurized wearing of UMCS which formed near completely closed space showed higher mean periocular humidity than condensation humidity. Though the higher periocular humidity provides greater tear retention effect, the spectacles inducing condensation without antifog material are not practical for daily wear. PMCS provided sufficiently high periocular humidity; however the measurements were always slightly lower than the condensation point. These results suggested that personalization of moisture chamber provides appropriate fit.There were some limitations to this study. The present study had small sample size and did not include patient group and clinical evaluation. Further investigation will be needed to determine the clinical effectiveness of PMCS, involving subjective symptoms and objective ocular surface evaluation in patients with dry eye disease with larger sample size.PMCS made by 3D printer provides appropriate fitness to form semiclosed humid chamber. PMCS increased absolute value of 36.39% periocular humidity without condensation as compared to the ambient humidity and demonstrated significantly higher performance than UMCS. The wearing of PMCS would be an effective method to provide highly enough periocular humidity in low humidity environment.


Urothelial carcinoma ranks the ninth among malignant cancers. We conducted this study to identify which patients could benefit more from the treatment of programmed death-1 (PD–1)/programmed death-ligand1 (PD–L1) inhibitors.We performed literature searches, combined data from qualified literature and performed comparative analyses on the effectiveness of anti-PD–1/PD–L1 antibodies in patients with different PD–L1 expression levels.We divided patients into three groups according to the percentages of PD–L1-positive cells, namely the low- PD-L1 (PD-L1 < 1%), the medium-PD-L1 (PD-L1 ≥ 1 and < 5%) and the high–PD–L1 (PD-L1 ≥ 5%) groups. We found that the high-PD-L1 group responded significantly better than other groups , while the latter two groups responded similarly  to both PD–1 and PD–L1 inhibitors. Furthermore, we found that the medium-PD–L1 and high-PD–L1 groups responded similarly to PD-1/ PD-L1 inhibitors , while the low-PD–L1 group responded better to PD-1 inhibitors than PD-L1 inhibitors .Our results suggest that PD–L1 positive patients should be defined as those with ≥ 5% or greaterPD-L1-positive cells. PD-1 antibodies performed better only in the low-group patients, likely because they could block the interactions of PD–1 with both PD–L1 and PD–L2. The incidence of urothelial carcinoma has become the ninth of all malignant tumors and the first in urinary tract cancers . It leadTumor microenvironment plays an important role in tumor immunity . ProgramIt has been shown in many clinical trials that cancer patients with higher expression of PD-L1 received better antitumor effect to PD-1/PD-L1 inhibitors , 11. SimWe retrieved in total 95 articles from public databases using the above-mentioned keywords. After reading abstracts and full texts, we kept six articles for further analysis; in total 828 participants were tested in the six studies. Please consult Figure Three out of the six studies evaluated PD-1 antibodies, of which two evaluated nivolumab , 20 and We first tested whether patients with higher-ratio of PD-L1-positive cells responded better to PD-1/PD-L1 antibodies. Either of 1% and 5% was often used in the literature to classify patients into “positive” and “negative” groups, according to whether there are more than 1% or 5% PD-L1-positive cells in a patient.P = 0.004, OR = 0.54,95%CI:0.39-0.85; see also Figure Of all the studies, 50 of the 345 (14.50%) patients with PD-L1 ≥ 1% level achieved objective responses, while 109 of 483 (22.57%) patients with PD-L1 < 1% level achieved objective response, confirming that patients with higher-ratio of PD-L1-positive cells indeed responded better to PD-1/PD-L1 antibodies  patients with PD-L1 ≥ 5% level achieved objective responses, as compared with 66 objective responses out of 481 (13.72%) patients with PD-L1 < 5% level; therefore patients with higher-ratio of PD-L1-positive cells responded better to PD-1/PD-L1 antibodies . In some studies, patients of the two groups were often combined; again, we found no significant differences in the combined datasets in the responses to PD-1 and PD-L1 antibodies.We next sought to compare the effectiveness of PD-1 and PD-L1 antibodies in patients with different PD-L1-positive cell ratios. In the literature, patients were often grouped into the following subgroups: PD-L1 < 1%, PD-L1 ≥ 1% but < 5%, and PD-L1 ≥ 5%; we referred them as to Low-PD-L1, Medium-PD-L1 and High-PD-L1 groups respectively in the following analysis. As shown in Figure P = 0.046, OR = 1.92, 95%CI: 0.98, 3.89; Fisher’s Exact Test) . However, patients in the Low- and Medium- groups responded similarly to both types of antibodies   Figure . These rIn this study, we performed a meta-analysis on the effectiveness of anti-PD-1/PD-L1 antibodies in urothelial carcinoma patients with different ratios of PD-L1-positive cells. We first confirmed previous findings that patients with higher ratios of PD-L1-positive cells responded significantly better to both PD-1 and PD-L1 antibodies than those with lower ratios of PD-L1-positive cells.We also checked whether patients responded better to one type of antibodies than the other. We found that in most cases, patients had similar ORRs between the two types of antibodies in the Medium-PD-L1 and High-PD-L1 groups. However, in patients with less than 1% PD-L1-positive cells (the Low-group), the objective response ratio was statistically better to PD-1 antibodies than to PD-L1 antibodies. This is likely that PD-1 antibodies are more sensitive to lower ratio of PD-L1-possitive cells, becausePD-1 antibodies block the interaction between PD-1 with PD-L1 and PD-L2, while PD-L1 antibodies only block the interaction between PD-1 with PD-L1 , 23. HowThe definition of PD-L1 “positive” patients is not very clear so far . CutoffsIn conclusion, PD-L1 positive patients of urothelial cancer response more than negative ones, and we suggest 5% or greater as the cutoff to distinguish them. PD-1 antibodies is better than PD-L1 antibodies in PD-L1 < 1% subgroup with urothelial carcinomas patients. Further clinical trials need to be performed to establish the position of immunotherapy in urothelial cancer.trials.gov and American Society of Clinical Oncology (ASCO) meeting abstracts for relevant articles published in English. All the articles were published before July, 2017. We used the following keywords to search for the articles: “PD-1”, “PD-L1” and “urothelial carcinoma”, “urothelial cancer”, “urothelial tumor” combined with “nivolumab”, “atezolizumab”, “pembrolizumab”, “avelumab”, “durvalumab”.We performed a systematic review search of literatures in Pubmed, Embase, clinical The articles must satisfy the following conditions: first, all patients suffering from advanced and metastatic urothelial carcinoma were treated with PD-1/PD-L1 antibodies. Second, certain types of raw data should be available in the original publications in order for us to combine results from multiple studies, including the total numbers of patients treated by PD-1/PD-L1 antibodies, the numbers of patients that responded to the treatment ), and the percentage of PD-L1-positive cells in the tumor microenvironment of these patients. In cases where the exact PD-L1-positive levels were unknown, patients should be grouped into sub-groups according to their rates of PD-L1-positive levels and the grouping criteria should be available.The data extracted from these articles was as below: the first author, the year of the article, the trial name, the antibody name, the different levels of PD-L1 expression and ORR (objective response rate), media progression-free survival (PFS), media overall survival (OS).The outcomes were ORRs and ORs of different PD-L1 levels in urothelial carcinoma patients by different antibodies.https://www.r-project.org) and R Studio (https://www.rstudio.com). Patients were grouped into subgroups according to the percentages of PD-L1-positive cells. The ORR value was calculated for each subgroup. Between-subgroup comparisons were performed by one-tailed Fisher-test using the Fisher. test (matrix) function in R; Odds ratio (OR), its 95% confidence interval (95%CI) and the P-value were recorded for each comparison. It was to consider statistically significant if P < 0.05.All analyses were performed using R 3.4.1 (


The expression of Programmed cell Death Ligand 1 (PD-L1) is observed in many malignant tumors and is associated with poor prognosis including Gastric Cancer (GC). The relationship between PD-L1 expression and prognosis, however, is controversial in GC. This paper purports to use a meta-analysis to investigate the relationship between PD-L1 expression and prognosis in GC. For this study, the following databases were searched for articles published from June 2003 until February 2017: PubMed, EBSCO, Web of Science and Cochrane Library. The baseline information extracted were: authors, year of publication, country where the study was performed, study design, sample size, follow-up time, baseline characteristics of the study population, pathologic data, overall survival (OS). A total of 15 eligible studies covering 3291 patients were selected for a meta-analysis based on specified inclusion and exclusion criteria. The analysis showed that the expression level of PD-L1 was associated with the overall survival in GC . In addition to the above, subgroup analysis showed that GC patients with deeper tumor infiltration, positive lymph-node metastasis, positive venous invasion, Epstein-Barr virus infection positive (EBV+), Microsatellite Instability (MSI) are more likely to expression PD-L1. The results of this meta-analysis suggest that GC patients, specifically EBV+ and MSI, may be prime candidates for PD-1 directed therapy. These findings support anti-PD-L1/PD-1 antibodies as a kind of immunotherapy which is promising for GC. Worldwide, gastric cancer (GC) is the fourth most common malignant disease in males  and the third leading cause of cancer mortality in males , especially in Eastern Asia , Central and Eastern Europe, and South America, and lowest in Northern America and most parts of Africa. DevelopRecently, some clinical trials have indicated that monoclonal antibodies that target PD-1 or its receptor PD-L1 prevent the inhibitory effects of PD-1/PD-L1 pathway and enhance T cell functions, leading to impressive outcomes in patients with cancers–9. HowevThrough a meta-analysis, this review focuses on PD-L1 expression and its association with clinical outcomes in GC. Furthermore, this research attempts to show that the potential of PD-L1 positive patient to obtain optimum treatment benefit, appears promising. It might pinpoint patients most likely to strongly benefit from the inhibition of PD-L1/PD-1 as monotherapy compared to those that may most likely require a different or combinatorial approach in GC.Studies indexed, from June 2003 until February 2017 were systematically searched in the following databases: PubMed, EBSCO, Web of Science and Cochrane Library. The search terms used were: (“stomach neoplasms” OR “gastric cancer” OR “advanced gastric cancer” OR “gastric carcinoma” OR “stomach cancers”) AND (“PD-1” OR “PD-L1” OR “programmed death 1” OR “programmed death ligand 1” OR “programmed cell death ligand 1” OR “programmed death 1 ligand 1” OR“B7-H1”). Additionally, the reference lists of the selected articles were manually reviewed to obtain other potentially relevant articles. Selected publications were all in the English language.From the potentially relevant articles obtained above, those that indicated correlation between prognosis  and PD-1/PD-L1 in GC were selected.For inclusion in this meta-analysis:Articles were limited to those dealing human subjects only.All patients with GC would have been diagnosed by pathological evidence.Expressions of PD-L1 would have been detected by Immunohistochemical (IHC) Assay from gastric carcinoma specimens.All patients had been followed up and results reported.The following criteria were used to exclude irrelevant papers:The literature was not the original article , or a literature duplication.The object of this study was cellular-based or animal-based.The study also covered other malignancies, or the study did not include the analysis of expressions of PD-L1 in subgroups.Data from each included study was independently extracted. The following baseline information was used: authors, year of publication, country where the study was performed, study design, sample size, follow-up time, cut-off criteria for overexpression (the definition of positive PD-L1), baseline characteristics of the study population, pathologic data, overall survival (OS).This meta-analysis was conducted in accordance with the guidelines of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) 2015 statement–13. The Hazard ratios (HR) including 95% Confidence interval (CI) were used to assess the association between PD-L1 expression level and OS in GC. An observed HR > 1 implied a worse prognostic significance for the group with elevated PD-L1 expression. Conversely, HR < 1 implied a worse prognostic significance for the group with decreased PD-L1 expression. Revman 5.3 Software  was used to evaluate heterogeneity between studies by Cochrane Q-test and P-values. Estimates of HR were weighted and pooled using the Mantel-Haenszel random effect model. The Stata 12.0 Software  was used to evaluate the sensitivity and publication bias of the studies. Publication bias was evaluated by Begg’s and Egger’s test, P < 0.05 was considered statistically significant. Begg’s and Egger’s test of publication bias was not performed on analysis subgroup with less than 5 studies because of low sensitivity of qualitative and quantitative tests.The search strategy originally generated 274 relevant clinical studies in English. Of these, 149 were eliminated because of repetition, 63 were excluded based on evaluation of their title or abstract. The remaining 62 articles were scrutinized by a full-text review. Based on the inclusion and exclusion criteria, 15 studies were selected for this meta-analysis. The detailed search and study selection process is shown in The 15 studies published between 2006 until 2016, included 3291 patients at baseline, with a maximum sample size of 451 and a minimum sample size of 96 participants. The majority of the studies were reported in Asia, 7 in China, 4 in Japan, 3 in South Korea, and 1 in Germany. The defining criterion for PD-L1 positive was that PD-L1 expression was observed in tumor cells by IHC. The discrepancies in the percentage of the PD-L1 positive expression patients were due to the variation in the cut-off values with the maximum ratio at 69.40% and the minimum at 14.32%. 1925(58.49%) patients did not undergo radiation or chemotherapy before surgery in 8 studies, others did not report. The characteristics of the included studies have been shown in A total of 15 studies reported that the expression level of PD-L1 was related to OS. 11 studies indicated that PD-L1 overexpression was associated with poor prognosis of GC, conversely, 3 studies reported that PD-L1 overexpression was associated with better prognosis, and there was no indicated associations 1 study. Analysis showed that the expression level of PD-L1was associated with the OS in GC  .As shown in Begg’s and Egger’s test was used to evaluate the publication bias, respectively. In this meta-analysis, Begg’s and Egger’s test indicated no publication bias among included articles regarding the HR and OS (Begg’s test: P = 0.276 and Egger’s test P = 0.061) . There weff) cells that are produced), the activated Teff cells can’t specifically recognize and bind to cancer cells, T cells therefore cannot kill their target cancer cells. In other words, some molecules, namely factors, may act to modulate activated anti-tumor T cell, such as PD-L1 Increased PD-L1 expression by tumor cells may induce and maintain iTreg cells in the periphery, thereby increasing the suppression of anti-tumor T cell responses and allowing tumor progression[As mentioned by: ‘Regulaeg) cells. PD-L1 iogression.In effect, this research paper tries to evaluate the association between the PD-L1 overexpression and the clinical pathological features. From this meta-analysis, gastric cancer patients with deeper tumor infiltration, positive lymph-node metastasis, positive venous invasion, E-B virus infection positive, MSI are more likely to express PD-L1. The results of subgroup analysis tend to support the idea that patients with positive PD-L1 have unfavorable prognosis.A recent study aiming tet al.[Beyond the differential presence of PD-L1+ cells in EBV+ and EBV- GC, Derks et al. observedTo the best of our knowledge, it is the first meta-analysis to investigate the association between PD-L1 expression and E-B virus infection and MSI in GC. This study shows that the expression level of PD-L1 is higher in EBV+ GC and MSI than in EBV- GC and Microsatellite stability (MSS). The result suggests that specifically EBV+ GC and MSI may be prime candidates for PD-1directed therapy.et al.[et al.[et al.[et al.[The limitations of this study should be stressed on. The heterogeneity among included studies cannot be ignored. We were unable to control for factors such as environmental conditions, racial differences, socioeconomic situation, postoperative treatment, follow-up, all of which are known to influence the OS. Besides, these studies have used a number of monoclonal and polyclonal PD-L1 antibodies for immunohistochemistry and a variety of different scoring schemes/criteria to define positive PD-L1 expression. For example, Eto et al. and Gengl.[et al. reportedl.[et al. and Bogel.[et al. considerl.[et al. to 69%[1l.[et al..Publication bias is another possible reason, wherein studies showing that PD-L1 was not a prognostic factor could be less likely to be published than studies showing that PD-L1 was a prognostic factor. Then the included studies were only English researches, no other languages. Beyond publication bias, the patients covered in this study were strictly from the countries where the studies were made. The conclusion would hence apply to those countries only unless the study is extended to other countries representatively. Perhaps the racial and social factors were also at play here as it has also been observed in other fields other than medicine. Their effects could have been cofounded with those being measured here. More representative samples of patient studies could be needed for stronger conclusion. Also, grey literature was not accounted for in this study as only studies published in the English language were chosen, 46.This meta-analysis was to firstly evaluate the association between the PD-L1 expression and a specific molecular subgroup (EBV+ and MSI) in GC. Furthermore, gastric cancer patient with EBV+ and MSI tend to show PD-L1 expression, which demonstrated that specifically EBV+ GC and MSI may be prime candidates for PD-L1 directed therapy. However, further large-scale and comprehensive researches are needed to support our results and conclusion.S1 Fig(A) sex, (B) age, (C) tumor site, (D) tumor size, (E) tumor differentiation, (F) Lauren Classification, (G) depth of infiltration,(H) lymph-node metastasis, (I) TNM stage, (J) lymphatic invasion, (K) venous invasion, (L) neural invasion, (M) E-B virus infection, (N)MSI-status.(TIF)Click here for additional data file.S2 Fig(A) Asian, (B-1) stages I-IV, (B-2) stages II-III, (B-3) stages I-III, (C) follow-up more than 5 years, (D-1) cut-off value >1%, (D-2) cut-off value >5%, (D-3) cut-off value >10%, (D-4) cut-off value >50%, (E-1) location within tumor cells were cytoplasmic, (E-2) location within tumor cells were cytoplasmic and nuclear, (F) no patients underwent pre-operative chemoradiotherapy, (G) patients underwent postoperative adjuvant Chemotherapy.(TIF)Click here for additional data file.S3 Fig(A) sex, (B) tumor site, (C) tumor size, (D) tumor differentiation, (E) depth of infiltration, (F) lymph-node metastasis, (G) TNM stage, (H) lymphatic invasion, (I) venous invasion.(TIF)Click here for additional data file.S4 Fig(A) sex, (B) tumor site, (C) tumor size, (D) tumor differentiation, (E) depth of infiltration, (F) lymph-node metastasis, (G) TNM stage, (H) lymphatic invasion, (I) venous invasion,(J)OS.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.