The improvements brought by the integration of graphene onto silicon photonic waveguides could bring us a step closer to realising compact all-optical control on a single chip.The hallmark of silicon photonics is in its low loss at the telecommunications wavelength, economic advantages and compatibility with CMOS design and fabrication processes. These advantages are however impeded by its relatively low Kerr coefficient that constrains the power and size scaling of nonlinear all-optical silicon photonic devices. Graphene, with its unprecedented high Kerr coefficient and uniquely thin-film structure, makes a good nonlinear material to be easily integrated onto all-optical silicon photonic waveguide devices. We study the design of all-optical graphene-on-silicon (GOS) waveguide modulators, and find the optimized performance of MW cm This is due to limits of metallic interconnects facing signal attenuation and large power consumption at higher data rates2. One of the solutions is to replace them with optical interconnects. The replacement candidate, silicon photonics, is viable given its potential low cost and high compatibility with CMOS design and manufacturing process3. Silicon photonics also interface well with electronic transistors through power-efficient optoelectronic transceivers. Recently, Sun et al. has successfully demonstrated a working prototype of a chip-scale electronic-photonic system based on the silicon photonics platform4.The thriving success of the electronics microprocessor over the past four decades is achieved with the abundant availability of silicon. However, in recent years, the increasing demand for faster processing speed and larger bandwidth have not been met, saturating at around 3 to 4 GHz5. In all-optical computing for silicon photonics, the nonlinear effects are used to achieve modulation. Nonlinear effects in silicon photonics have been demonstrated to process optical signals at speeds of beyond 100 Gbit/s6. Besides processing optical signals, nonlinear effects are also used for sensing and generation of photons for lasing and amplification. However, the inherent nonlinear Kerr effects of silicon is of the low range of 6 × 10−18 m2 W−1. To achieve a reasonable level of contrast needed for optical modulation, nonlinear optical devices often need to be operated at high optical intensities and long optical device lengths. This results in high power consumption and large device footprints, which runs in contrary to the original aim to scale down device size and energy consumption in computing chips7.Current research is also pushing for elimination of electronic transistors for seamless integration of an all-optical computing platform−7 to 10−13 m2 W−1 15. Being a two-dimensional atomically thin-film material, graphene alone is not suitable to be used as photonic waveguide due to its poor optical confinement. There are efforts to design around the thin-film nature of graphene by implementing the nonlinear optical devices on graphene plasmonic waveguides21; however, due to their short propagation length of only a few micrometers23, they are not compatible with the longer-ranged photonics, only suitable to be used in an all-plasmonics platform.The best way to mitigate the disadvantages of silicon nonlinear photonics is to integrate them with novel high Kerr-coefficient materials while keeping the silicon platform for its economic advantages. One of the best candidate material is graphene, which has a high Kerr-coefficient from 1024, with a conclusion that graphene’s nonlinear performance is ordinary due to its high losses. However, in this paper, we show that if we take into account the nonlinear reduction in losses, or even designing extinction modulators, the performance of graphene may exceed that of silicon-on-insulator (SOI) waveguides.Prior papers discussed graphene-based modulators placed on dielectrics only in the context of its real refractive index changesHence, by integrating graphene onto silicon waveguides, we simultaneously make use of the photonic confinement and long-range waveguiding properties of silicon-based waveguides while leveraging the high optical nonlinearities of graphene for optical switching. In this paper, we will study in detail how to integrate graphene onto a silicon waveguide and the corresponding performance improvements in all-optical switching on silicon photonic waveguides.2 with refractive index of 1.44. A graphene sheet with atomic thickness of ~0.3 nm is then laid on top of the waveguide, with a sheet width large enough to cover the fundamental mode-area of the silicon rib. Our choice of adopting the SOI rib waveguide is based on extensive studies on its good confinement and low waveguide propagation and bending losses26. On top of that, this structure exposes the top waveguide surface, which allows easy placement of graphene close to the mode-propagation area, allowing graphene to have maximum interaction with the fundamental optical mode. To control nonlinear optical response of the modulator, an optical pump is shined directly on the graphene surface to modulate the Kerr refractive index.Figure Ageff, which is written asTo quantify the interaction of the waveguide mode with the graphene sheet, we define the effective mode area of graphene, Ageff are easily found with the help of mode simulations in COMSOL. Here, we shall limit our study to only the fundamental TE and TM modes of the silicon rib waveguide. Figure Ageff of the TE and TM modes vary with wavelength in the range from 1.3 to 1.7 µm. The Ageff of the TE mode is relatively constant at around 1.2 × 103 µm2. On the other hand, the Ageff of the TM mode rises significantly with the increase in wavelength. A closer inspection on the electric-fields on the TM mode reveals their asymmetrical distribution at the top and bottom interfaces of the waveguide. At longer wavelengths, the electric-fields are increasingly distributed more to the bottom of the waveguide, thus the mode interacts less with the graphene sheet.Solutions of 27ω, relaxation frequencies ν1 and ν228, and Fermi-level EF at room temperature T = 300 K. The optical conductivity can be used to derive the refractive index of graphene usingdeff is the graphene’s layer thickness approximated to 0.3 nm. The refractive index of graphene is plotted with wavelength in Fig. EF < 0.4 eV), graphene is a lossy dielectric in the wavelength range from 1.3–1.7 µm. Graphene-on-substrate has a natural substrate doping of around 0.1–0.2 eV, thus we will base our device analysis around this range of Fermi levels29.The linear graphene optical conductivity is described by the Kubo formula−11–10−13 m2W−1 around 1.55 µm15. Graphene’s nonlinear conductivity is calculated using semiconductor Bloch equations based on graphene’s tight-binding model by Cheng et al.15. These values can be transformed to the nonlinear refractive index using30ngraphene and kgraphene are the real and imaginary linear refractive indices of graphene, and EF as shown in Fig. EF from 0.1–0.25 eV, much unlike the behavior in the midinfrared where the low EF regime has nonlinearities higher by a few orders21.This can be qualitative explained by the fact that at low-frequency regime the optical response is dominantly contributed by intraband process which has a stronger Fermi level and frequency dependence while at high-frequency regime the dominating interband process has a much weaker frequency and Fermi level dependences13. At 1.55 µm, the Kerr coefficient of graphene is between 2–5 × 10−11 m2W−1, which is seven orders higher than silicon’s Kerr coefficient of 6 × 10−18 m2 W−1.Meanwhile, the nonlinear optical conductivity of graphene has been obtained from theory and experiments, which ranges from 1028, is to lower the overall propagation loss of graphene after modulation. This would augment the phase-modulation performance of the graphene-on-silicon waveguide, and at the same time enable the design of an extinction-based modulator.It is noted that the magnitude of graphene’s real and imaginary Kerr coefficients is negative. The impact of the negative imaginary Kerr coefficient, also called the saturable lossFinally, we also take into account the saturation intensity of the nonlinear Kerr modulation using the standard experimental definition−2 as shown in Fig. The saturation intensities are in the range of a few MW cmΔn and Δk, would give us the effective nonlinear refractive index throughTo obtain the effective nonlinear modulation of the GOS waveguide would require analysis of the entire optical mode propagation in the waveguide structure. This is easily achieved through mode simulations in COMSOL, where the effective refractive indices before and after the optical modulation are analysed. In order to give a more accurate picture of the nonlinear performance contributed by graphene, we have omitted the nonlinear refractive index of silicon from our simulations. The difference between the values, n and k denote the effective waveguide indices instead of the material indices. Also, the optical intensity, I, has the implicit term of n2 and k2 values, we use I = 1 kW cm−2 in the simulation, which is significantly less than Isat.Here, it is noted that n2 and k2 for both the fundamental TE and TM modes. The slight increase of the waveguide nonlinear indices with wavelength for the TE mode is in line with trend for the nonlinear material indices for graphene. For the TM mode, however, the waveguide nonlinear indices drop off at the longer wavelengths due to diminished optical interaction with graphene, which arose from the asymmetric distribution of the electric-fields as has been shown before in Fig. The linear effective waveguide indices of the GOS waveguide are shown in Fig. k2 is at least an order higher than that of n2, even though the material n2 and k2 magnitudes are almost the same. This is intuitively understood by looking at the linear material properties of both silicon and graphene. The refractive index of silicon is large (nSi = 3.48) and since the optical mode occupies the silicon to a large spatial extent, graphene’s nonlinear change in material refractive index have little influence on the overall effective waveguide refractive index. This is not the case for the extinction coefficient, as while it is negligible for silicon in the 1.3–1.7 µm spectrum, it is quite substantial for graphene (kgraphene ~3). Hence, any nonlinear change in graphene’s extinction coefficient would show up prominently in the effective waveguide losses. The implication of this result is that the GOS nonlinear waveguide is better suited to be designed as an extinction-based modulator, as we will understand better through further analysis below.Another important point of observation is that the magnitude of the waveguide λ0 is the free-space wavelength. We can also define the nonlinear parameter containing the nonlinear extinction coefficient through modifying the expression toAnother way to quantify the nonlinearity of a waveguide that also takes into account the effective optical mode area is through its nonlinear parameters. The common definition of the nonlinear parameter containing the real nonlinear index is given as−1 m−1, are plotted in Fig. γn is expected to be low since the optical mode interaction with graphene is minute. In Fig. −1 m−1 for the TE mode and an average of 3–10 W−1 m−1 for the TM mode, which is far lower than the typical values for SOI waveguides in the order of 100 W−1m−1. However, an interesting case is observed for γk, which has values of more than two-orders higher than its real counterpart, in the range of 150–400 W−1 m−1 (or 650–1700 dB W−1 m−1) for the TE mode and an average of 300–1000 W−1 m−1 (or 1300–4300 dB W−1 m−1) for the TM mode, as shown in Fig. The nonlinear parameters, both in units WHere we shall study the construct of two types of nonlinear GOS modulator, which are the phase and extinction modulators respectively. For phase modulator, the objective is to perform a π-phase shift for constructive/destructive interference in a Mach-Zehnder interferometer (MZI), so as to achieve maximum contrast between the on/off states. It is also assumed that only one arm of the MZI is covered with graphene to minimize the device’s insertion loss. The maximum phase-shift of such a modulator is given asI implicitly carries the usual saturation terms, while the nonlinear effective length of the waveguide will lengthen according to the optical pump intensity due to the saturable absorption effect. The use of the nonlinear effective waveguide length ensures that the output signal intensity from the GOS waveguide is high enough to combine interferometrically with the signal from the reference arm.Here, On the other hand, for the extinction modulator we can similarly write the maximum extinction change asEF = 0.1 eV and operating wavelength of 1.55 µm. For an MZI phase modulator, the optical signal is split equally to both arms, and only one of the arms is coated with the graphene layer. To determine the minimum optical pump intensity required for the device to perform a π-phase shift, we plot out ∆ϕmax in Fig. I ~ 0.3 GWcm−2, for both the TE and TM modes. The corresponding nonlinear effective waveguide length for these two modes, read from the Leff-nonlinear plot in Fig. To illustrate the design steps of the nonlinear modulators, we first choose an example GOS waveguide, with parameters of graphene ∆αmax in Fig. −2, and the corresponding Leff-nonlinear for the TE and TM modes are 0.22 mm and 0.14 mm respectively, which are at least 70 times shorter than the phase modulator. To accommodate switching of both modes, we pick the shorter waveguide of the two (0.14 mm) to minimize insertion loss for the TM mode. Finally, Fig. −2 pump intensity, the TE mode switches from −8 dB to −3 dB, representing a contrast of 5 dB, while the TM mode switches from −12 dB to −4 dB, a contrast of 8 dB. There is also the possibility of constructing even shorter waveguides by allowing an increase to the pump intensity.On the other hand, the design of the extinction modulator is more straightforward. In a similar fashion, we plot −18 m2 W−1, and Aeff of 2 × 10−13 m2 for the fundamental TE mode, which transcribes to a nonlinear parameter of ~100 W−1 m−1 at the telecommunications wavelength7. Using a waveguide length of 1.5 cm, the required optical intensity to modulate a π-phase shift is 0.86 GW cm−2. The nonlinear GOS phase modulator, in comparison, require a slightly lower optical intensity at 0.3 GW cm−2.A quick performance evaluation could be made for the nonlinear GOS waveguide devices against a typical nonlinear SOI waveguide. The typical 500 nm × 200 nm SOI waveguide have a nonlinear refractive index of 6 × 10−2.In stark contrast to the nonlinear GOS phase modulator, the nonlinear GOS extinction modulator compares very favorably to the typical nonlinear SOI modulator. The nonlinear GOS extinction modulator has a device length 70 times shorter at 0.14 mm and the required optical pump intensity is 80 times lower at 10 MW cm−2), and the saturable absorption phenomenon dominates. However, for higher optical intensities above 10 GW cm−2, it is possible that the two-photon absorption (TPA) process will take over and cause an increase in device optical loss with increasing optical intensity, as evidenced in ref.31. Also, the temporal optical response of graphene is ultrafast, possibly reaching Petahertz (PHz) or sub-femtosecond (sub-fs) timescales from a very recent study32.Throughout the discussion of our simulated results, it is assumed that the optical intensity is low  in direct contact with graphene. Fabrication of such structures may be possible in the near future with advancement of technologies such as wafer transfer and embedding of grapheneWe have studied the use of graphene to enhance the nonlinear properties of an SOI photonic waveguide. Graphene has a giant nonlinear Kerr coefficient which enables all-optical modulation at low optical intensities. Also, its existence as a thin film makes it easily integrated onto current existing SOI waveguide platforms, which is a highly desirable structural property, in contrast to other bulk nonlinear materials where integration requires etching and deposition, or hybrid integration via evanescent coupling to a separate waveguide platform.−2. Direct irradiation of the optical pump on the graphene surface requires very low optical intensities to perform modulation with very high switching contrast of at least 5–8 dB. In addition, GOS waveguide lengths are by far shorter than standard SOI nonlinear waveguides. These performance improvements brought by the integration of graphene on nonlinear silicon photonic waveguides could pave the way for more compact and low-powered all-optical devices for chip-scale integration.The GOS waveguide’s only drawbacks are the high linear absorption and limited access to the photonic waveguide modes. Nonetheless, the high linear absorption of graphene has already been compensated by its high saturable absorption at very low optical intensities in the order of 10 MW cmI = 1 kW cm−2, is separately calculated using data from refs15. The calculated value is added to the linear refractive index for simulation. The difference between the effective indices from the two sets of simulation, neff1 and neff2, would give us the effective index changeTo obtain the effective nonlinear waveguide Kerr index, two sets of effective waveguide indices are obtained using two-dimensional MODE simulations in COMSOL application. In the first set, graphene’s linear refractive index is used in the simulation. In the second set, graphene’s nonlinear change in refractive index for an optical pump intensity, The effective nonlinear waveguide Kerr index is then easily found usingAll data generated or analyzed during this study are included in this published article.


Cancer therapy has been constantly evolving with the hope of finding the most effective agents with the least toxic effects to eradicate tumors. Cancer immunotherapy is currently among the most promising options, fulfilling this hope in a wide range of tumors. Immunotherapy aims to activate immunity to fight cancer in a very specific and targeted manner; however, some abnormal immune reactions known as immune-related adverse events (IRAEs) might occur. Therefore, many researchers are aiming to define the most proper protocols for managing these complications without interfering with the anticancer effect. One of these targeted approaches is the inhibition of the interaction between the checkpoint protein, programmed death-receptor 1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), via a class of antibodies known as PD-1/PD-L1 inhibitors. These antibodies achieved prodigious success in a wide range of malignancies, including those where optimal treatment is not yet fully identified. In this review, we have critically explored and discussed the outcome of the latest PD-1 and PD-L1 inhibitor studies in different malignancies compared to standard chemotherapeutic alternatives with a special focus on the clinical efficacy and safety. The approval of the clinical applications of nivolumab, pembrolizumab, atezolizumab, avelumab, and durvalumab in the last few years clearly highlights the hopeful future of PD-1/PD-L1 inhibitors for cancer patients. These promising results of PD-1/PD-L1 inhibitors have encouraged many ongoing preclinical and clinical trials to explore the extent of antitumor activity, clinical efficacy and safety as well as to extend their applications. Cancer therapy has regularly shown glimmers of hope in the search for the most effective measure with the least toxic effects to eradicate tumors. Various approaches have been implemented in cancer therapy, which include surgery, radiation, chemotherapy and targeted therapy. The continuous process of research has shifted the focus in the last century toward immunotherapy as a novel tool for fighting cancer. The concept of utilizing immunity as a therapeutic technique was first introduced in 1957 by Thomas and Burnett, who discussed the ability of tumors to provoke an immune response ,3. Thus,The growing evidence on the role of immunity in treating cancer has given rise to multiple strategies such as the inhibition of cancer checkpoint proteins. Checkpoint proteins are considered a vital part of the immune system expressed on different immune cells with the ability to initiate an immune response either by their activation or inhibition . In 1997The activation of checkpoint proteins expressed on T-lymphocytes such as cytotoxic T-lymphocyte antigen 4 (CTLA-4) and PD-1, usually helps the tumor cells to escape detection by the immune system. Thus, the inhibition of these checkpoint proteins would activate the immune system against tumors . Compare+ cytotoxic T cells from inducing an anti-tumor response. Thus, tumors would acquire an adaptive resistance through PD-L1 upregulation, which is one of the obstacles against the application of PD-1/PD-L1 inhibitors .,25.23,25Other studies were performed to explore the effect of nivolumab in NSCLC treatment. Some of these trials assessed its efficacy as a second-line treatment upon the initiation of other types of therapy. The latest related preformed trials are the CheckMate 017 and CheckMate 57 ,29. BothThese findings were the reasons behind the FDA approval of nivolumab use as a second-line treatment in advanced NSCLC, in 2015 . FurtherPembrolizumab MK-3475) is an IgG-1 mAb that is considered to be a highly potent and selective PD-1 inhibitor ; it was 75 is an The efficacy and safety of pembrolizumab as a first-line agent in NSCLC gave more promising results than nivolumab against chemotherapy. The ORR with pembrolizumab as a first-line therapy was 44.8%. However, the ORR of the docetaxel-treated group was only 27.8% with 2.2 months as the median time of response to both groups . StudiesThe antitumor efficacy of pembrolizumab according to the results of the KEYNOTE-012 study was durable; it revealed a good response rate. About 33 urothelial cancer patients were enrolled in this phase IB study to receive pembrolizumab at a dose of 10 mg/kg every two weeks. The retrieved ORR was 38% among the PD-L1-positive patients and 5% of the pembrolizumab-treated patients displayed grade 3–4 adverse effects .Currently, a phase II study (KEYNOTE-052) is running, in which urothelial cancer patients are enrolled to receive pembrolizumab as a first-line agent . If thisMM is another FDA approved indication of pembrolizumab as an anticancer agent. In KEYNOTE-002, a phase II clinical trial , pembrolFurthermore, the efficacy of pembrolizumab in MM patients was demonstrated in another large phase I clinical trial, which enrolled MM patients to receive pembrolizumab at a dose of 2 mg/kg or 10 mg/kg once every two or three weeks . The higA similar phase IB study (KEYNOTE-028), which was performed in PD-L1-positive ovarian cancer patients, provided promising preliminary results. Among the 26 enrolled patients, one patient achieved a complete response and two patients showed partial response after treatment with 10 mg/kg of pembrolizumab once every two weeks. The best OR was 11.5% and all patients experienced ≥1 adverse events such as fatigue, anemia and decreased appetite . Thus, fPembrolizumab applications suggest certain similarities to nivolumab-reported studies and findings. Both agents share comparable results in terms of their clinical efficacy in certain tumor types such as in melanoma, in addition to the fact that both were the first FDA approved checkpoint inhibitors for the same indications . HoweverPidilizumab (CT-011) is an IgG-1 mAb that targets PD-1 protein . The effBesides pidilizumab activity in hematological malignancies, a phase II clinical trial revealed its powerful activity in refractory cases of melanoma . In thisAMP-224 is a fusion protein composed of the programmed death-ligand 2 (PD-L2) extracellular region and the fragment crystallizable (FC) region of the human IgG-2A  63]. Un. Un63]. Therefore, AMP-224 might exhibit distinct safety and efficacy results as an anticancer agent compared to other PD-1 inhibitors, due to the differences in their biological nature and mechanisms of action. An ongoing study in solid tumors has shown promising tolerance in one patient to a dose of 30 mg/kg. Thus, more crucial outcomes are expected to be revealed by the end of the study . Other rAmong the specific reactions seen with the use of AMP-224 was infusion reactions, which can be explained by the unique nature of this agent . This phAMP-514 is another PD-L2 fusion protein that aims to activate immune defense cells by blocking PD-1 . This agAtezolizumab (MPDL3280A/RG7446) is an IgG-1 humanized antibody that blocks the PD-1/PD-L1 interaction, by targeting the expressed PD-L1 on numerous kinds of malignant cells . The bloIn 2016, atezolizumab became the first agent to gain FDA approval as a second-line treatment of urothelial cancer. This approval was based on the results of a cohort study that enrolled 310 urothelial cancer patients, previously treated with chemotherapy . Atezolip = 0.04) . Am. Am82]. 6 is a huMoreover, durvalumab was extensively studied in urothelial cancer. A phase IB study reported its efficacy in advanced urothelial bladder cancer patients with an ORR of 31%. Furthermore, a good safety profile was attained in this trial as the most reported side effects were fatigue and decreased appetite and only 4% of these patients showed grade 3 treatment-related adverse events . These pThe continued success of this agent encouraged the researchers to explore its efficacy and potential anti-cancer activity in several treatment combinations, aiming to come up with the optimum cure. However, the safety of this agent is still not fully guaranteed as its combination with other agents increased the potential toxicity. For example, an administered combination of durvalumab with a CTLA-4 inhibitor, tremelimumab, resulted in inflammatory myopathy in a 68-year-old NSCLC patient; fortunately, this adverse effect was resolved upon the discontinuation of this regimen .Avelumab (MSB0010718C) is a distinguished IgG-1 antibody that inhibits PD-L1 . It diffThe JAVELIN solid tumor studies include a phase IB trial in metastatic urothelial cancer (MUC), which showed impressive outcomes. Avelumab was administered as a second-line treatment to 44 MUC patients and resulted in an ORR of 40%, in addition to 12 weeks PFS of 70%. Similar to durvalumab, these positive findings in the management of MUC by avelumab pushed the FDA to accelerate its approval as a second-line treatment .The most exciting fact revealed about avelumab is its astonishing efficacy in a very aggressive immunogenic skin cancer type called mMCC. In March 2017, avelumab succeeded in obtaining FDA approval as an anti-cancer agent to cure mMCC . The JAVBeyond any doubt, avelumab is one of the unique PD-L1 inhibitors that tapered difficult malignancies such as mMMC, and it gave hope to mMMC patients after the absence of approved treatments for a long period. Most of the research on avelumab is still in progress, just like other PD-1/PD-Ll agents; the complete picture of this promising agent is yet to be presented as it might be the answer to many more pressing problems in the oncology field.Targeting PD-1/PD-L1 axes in an attempt to eradicate malignancies, via the activation of the immune system, demonstrated great success in numerous types of tumors. To date, the cumulative research efforts lead to the use of PD-1/PD-L1 inhibitors as official FDA approved agents in many tumors such as MUC, NSCLC, MM and MCC. In fact, the clinical field is very active nowadays, running trials to further evaluate the safety and efficacy of these agents . It is nWhen it comes to comparing PD-1/PD-L1 inhibitors to classical chemotherapeutic agents, it is clearly observed that some of them can be considered as equals. This was revealed in multiple reported studies of PD-1/PD-L1 inhibitors such as nivolumab and pembrolizumab, which presented similar, and in some cases even higher, responses regarding the potency in addition to being safer in different malignancies compared to standard chemotherapeutic agents ,53. HoweIn addition, the use of PD-1/PD-L1 agents is not as well established as the detailed defined use of chemotherapy. In fact, many trials are currently running with the aim of identifying the safest dosage regimens and establishing the most potent and proper combination of the PD-1/PD-L1 inhibitors with chemotherapy or any other known treatment for cancer. These combinations could be a therapeutic option with outstanding results, but the low safety might be the limiting factor, which is an obstacle that many trials are trying to solve. Another drawback against the application of these antibodies is the incidence of IRAEs, which can lead to fatal events on rare occasions and so cannot guarantee the safety of the patients.The low expression of PD-L1 in cancer cells is another limitation for the use of PD-1/PD-L1 inhibitors due to the direct correlation between the expression level of PD-L1 and therapeutic response. Therefore, the lower the level of PD-L1, the less the expected therapeutic outcome. Many studies have shown that the patient’s response to these agents depends on the level of the PD-L1 expression. This fact was illustrated by collective reports which showed that out of 1400 essayed patients, response was noted in 45% of the PD-L1-positive patients, while in the PD-L1-negative patients, only 15% showed a similar response. Hence, this offers certain selectivity regarding the responsiveness of patients to the use of PD-1/PD-L1 inhibitors ,90.Since these agents target immunity, they may result in abnormal immune reactions and generate toxicity that affects the skin, gut, lung, liver and other tissues . Among tIn conclusion, based on the reported basic and translational studies, the continuous research and development of PD-1/PD-L1 inhibitors has fostered breakthrough findings in the treatment of challenging tumors. Considering the safety of these agents, PD-1/PD-L1 inhibitors are generally better tolerated than classical chemotherapeutic agents. Nevertheless, more preclinical and clinical investigations are required to elucidate the key mechanisms and predictive biomarkers of the efficacy and safety of these agents and to limit the risk of IRAEs.


The role of hydrochloric acid treatment on the exfoliation result was carefully examined. The exfoliated products were characterized by X-ray diffraction (XRD) patterns, scanning electron microscopy (SEM), atomic force microscopy (AFM), and UV-Vis spectroscopy. The exfoliated g-C3N4 nanosheets exhibited a uniform thickness of about 3–5 nm and a lateral size of about 1–2 µm. A g-C3N4/graphene nanocomposite was prepared via a self-assembly process and was demonstrated to be a promising sensing material for detecting nitrogen dioxide gas at room temperature. The nanocomposite sensor exhibited better recovery as well as two-times the response compared to pure graphene sensor. The detailed sensing mechanism was then proposed.Graphitic carbon nitride (g-C Gas sensors are devices that able to respond to specific gasses and they play an important role in industrial chemical processing, environmental monitoring, agriculture, medicine, public safety, and indoor air quality control . Up to nRecent studies have revealed that two-dimensional (2D) materials, especially graphene, have the potential to detect numerous gases at room temperature owing to their large surface area, high carrier mobility, and low noise. However, many reports have also demonstrated that pristine graphene has a poor gas sensing property owing to the absence of dangling bonds in the structure ,9. In or3N4), as another emerging component with a two-dimensional characteristic structure has attracted considerable attention recently, due to the tri-s-triazine units that are connected by amino groups in each layer and the weak van der Waals forces between layers. The presence of nitrogen atoms in the graphene-like layered g-C3N4 structure gives g-C3N4 its unique properties, such as semiconductor properties, solid alkalinity, and complexing ability, thus endowing it with better performance than graphene in some aspects [3N4 has been extensively used as a polymeric photo-catalyst for solar hydrogen production and environmental purification, as well as oxygen reduction and evolution [3N4 exhibits excellent gas sensing properties owing to its excellent catalytic property [3N4 and graphene, a synergetic effect in sensing performance could be expected if the two components are composited as a gas sensor material.On the other hand, graphitic carbon nitride  or nitric acid (HNO3) was adopted to intercalate the acid ions into the interlayer space of g-C3N4 followed by an ultrasonic treatment, and ultrathin g-C3N4 nanosheets were successfully synthesized [3N4. Moreover, in these previous studies, the controlling factors in the exfoliation process, such as acid treatment time, were rarely considered.Up to now, the lack of a simple approach to producing 2D nanomaterials at a large scale has been the main obstacle to achieving wide application of 2D graphene-like materials. The feasibility of the recently developed ultrasound-assisted liquid-phase exfoliation method has been demonstrated for producing ultrathin nanosheets from bulk 2D material. The cavitation effect induced by the collapse of ultrasonic-produced bubbles generates liquid jets with high temperature, pressure, and speed, which would overcome the interlayer van der Waals force and break up the layered structure to yield individual nanosheets. However, this method is still not robust enough owing to the low yield and re-aggregation tendency after exfoliation. To address these issues, a possible route is the optimization of the solvent. It is reported that the maximization of exfoliation efficiency is related to the surface energy of the solvent. The comparative surface energy between the solvent and the 2D structured solute is favorable for the delamination. Xie and her co-researchers revealed that the surface energy of water matches well with that of g-Cin water . Anotherthesized ,28. Howe3N4 and g-C3N4 products exfoliated with different HCl acid treatment times were examined by X-ray diffraction (XRD) patterns. As shown in 3N4 and the resultant g-C3N4 products remain substantially the same except for some minor changes. No obvious difference was observed between the exfoliated products. All the samples presented a characteristic peak near 27.7°, which can be indexed to the (002) facet caused by the interlayer stacking reflection of conjugated aromatic systems. It was found that the (002) peaks of exfoliated g-C3N4 presented a slight left shift from 27.7° to 27.5°, suggesting loose packing in the exfoliated g-C3N4 product. It is worth noting that the intensity of the (002) peak decreased significantly in the exfoliated sample compared with the bulk sample, demonstrating the successful exfoliation of bulk g-C3N4. It was also observed that the sensitivity of the diffraction peak at 12.74°, which represents the period tri-s-triazine group, was reduced in the exfoliated g-C3N4 compared with the bulk g-C3N4. This could be attributed to the downgrade of the planar size after exfoliation.The crystal structures of bulk g-C3N4 displayed photoabsorption from ultraviolet to visible light. The bulk g-C3N4 has a band edge at approximately 460 nm corresponding to 2.70 eV. For comparison, all the exfoliated g-C3N4 samples have a band edge at approximately 450 nm, corresponding to a bandgap of 2.74 eV, indicating a slight blue shift of 0.04 eV. The increase of bandgap after exfoliation can be attributed to the well-known quantum confinement effect. When the thickness of the g-C3N4 product after exfoliation becomes ultrathin, approaching the dimension of the de Broglie wavelength, the motion of electrons is confined, leading to the splitting of energy levels, and thus the conduction and valence bands shift in opposite directions, resulting in the increase of the bandgap energy.The light absorbance of the samples was studied by UV-Vis spectroscopy. As shown in 3N4 products. According to the Lambert-Beer law of A/l = αC, where α is absorption coefficient, A is the absorbance, l is light path length, and C is the concentration of solute, the absorption of light is directly proportional to both the concentration of the solute and the thickness of the medium in the light path. Given that the light path was fixed throughout the measurement, the concentration of g-C3N4 could be estimated from the intensity of absorption. As observed from 3N4 product with 0.5 h acid treatment exhibited the lowest concentration. The concentration increased considerably as the acid treatment time extended to 1 h, and then slightly increased as the time further increased to 2 h, suggesting the exfoliation approached saturation.The UV-Vis absorption spectra of the samples also provide information of the concentration of the g-C3N4 with 1 h HCl acid treatment was investigated using atomic force microscopy (AFM). As shown in 3N4 nanostructure was observed. The thickness of the g-C3N4 nanosheet was estimated to be about 4 nm . 3N4, respectively. A large number of loosely stacked graphene nanosheets could be observed, suggesting successful exfoliation. On the other hand, the exfoliated g-C3N4 nanosheets exhibited a severe restacking, whereby it is difficult to identify an individual nanosheet. In the case of the composite, as shown in 3N4/graphene film was observed. Remarkably, the restacking phenomenon of g-C3N4 was greatly alleviated, which could be attributed to the self-assembly process between g-C3N4 and graphene [3N4/graphene composite.The microstructures of the graphene and g-Cgraphene . This fi3N4/graphene composite, respectively. The elemental mapping analysis, as shown in 3N4 [Energy-dispersive X-ray spectroscopy (EDS) analysis and elemental mapping were also utilized to investigate the elemental composition and distribution of the g-Cn in 3N4 .3N4/graphene nanocomposite, as displayed in 2O [2 C=C, respectively [2 hybridized aromatic N bonded to carbon atoms (C=N–C) existed in g-C3N4, which was known as the reactive pyridine N. Besides, the surface N/C ratio in nanocomposite was determined to be 0.27, which is closed to that in EDS analysis. The above XPS results further demonstrated the formation of g-C3N4/graphene nanocomposite.X-ray photoelectron spectroscopy (XPS) measurement was carried out to further study the chemical composition and element binding energies of g-Ced in 2O ,31,32. Tectively ,29. No C3N4/graphene composite gas sensor was examined with NO2 gas. The pure graphene sensor as a control sample was also tested simultaneously. 2 at room temperature. As observed, upon exposure to NO2 gas, the resistance of both sensors decreased, suggesting a p-type behavior. It is known that NO2 as a typical electron withdrawer would increase the hole concentration of the sensor materials, and thus decrease (or increase) the resistance of p-type (or n-type) semiconductors. Remarkably, the composite sensor exhibited two-times the response of pure graphene, indicating that the incorporation of g-C3N4 would benefit the sensing performance of the graphene sensor.The performance of the g-C2 gas was removed, while about 20% recovery was observed in the composite sensor within 200 s. This is possibly attributed to the thermal excitation of g-C3N4 that introduced active carriers and facilitated the desorption process of NO2 gas molecules. Our results indicate that the incorporation of specific semiconductors can achieve full recovery of the graphene sensor without the need for a complicated heating or vacuum process.It is well-known that intrinsic graphene shows poor recovery, partially due to the strong adsorption energy of the gas molecule. Thus, heating at an elevated temperature or vacuum treatments are required to clean the graphene sensor ,34,35. A3N4/graphene composite sensors toward NO2 ranging from 5 to 200 ppm. The sensors responded well with the target gas. The responses increased monodirectionally as the concentration of NO2 increased. No obvious saturation state was observed in the composite sensor, suggesting a broad detection range. Remarkably, the composite sensor showed two-times more response than the other sensors to pure graphene in all the tests. The stability property of our nanocomposite sensor was also examined as shown in 2, suggesting an excellent repeatability.3N4/graphene composite sensor is proposed, as schematically shown 3N4 nanosheet behaves similarly to a base that is ready to interact with the oxidized NO2 gas molecules. This characteristic of g-C3N4 nanosheet as well as its excellent catalytic property endows it with a promising capacity for the adsorption of NO2 molecules. On the other hand, graphene with superior carrier mobility would act similarly to a signal highway in the sensing process. Upon adsorption, the oxidized NO2 gas molecule with strong electron affinity deposits a hole in the g-C3N4 nanosheet. These hole carriers are then rapidly conducted by the graphene. In short, the complementary natures of g-C3N4 and graphene in the sensing process introduce a synergetic effect that contributes to the excellent sensing property of the nanocomposite sensor. In addition, considering the different roles of both materials in the sensing process, a tradeoff on the sensing performance would be expected if the composition of the composite is changed. Through systematic investigation, an optimized ratio of g-C3N4 and graphene could be achieved to obtain a high-performance sensor with excellent sensitivity and promising recovery property. This is the expected direction in the future research.The sensing mechanism of the g-CRaw melamine purchased from Sigma-Aldrich Korea Ltd.  was used as received. The fabrication process was modified from the reported methods ,37,38. T3N4 powder was added to 25 mL hydrochloric acid  and stirred for 1 h. The obtained transparent yellow dispersion was then filtrated and washed repeatedly with water until the pH value became neutral, followed by a drying process at 100 °C for 6 h. This acid-treated powder was re-dispersed in 200 mL deionized (DI) water, followed by sonication treatment for 2 h in an ultrasonic bath . Subsequently, the mixture was centrifuged at 8000 rpm for 10 min to remove the residual un-exfoliated g-C3N4 particles. Finally, a light white suspension was obtained. In order to determine the concentration of the product, 100 mL of g-C3N4 dispersion was transferred to a pre-weighted vial and dried in an oven at 100 °C for 24 h. The concentration of g-C3N4 in dispersion after 1 h acid treatment was determined to be about 50 μg/mL.In a typical synthesis, 0.5 g of yellow bulk g-CGraphene was prepared following the process developed in our previous work . Briefly3N4 and graphene were mixed with ultrasonic treatment for 1 h to achieve complete mixing. After mixing, the mixture was evaporated in an oven for 24 h at a moderate temperature (~60 °C) to improve the concentration of solute without introducing severe aggregation. Alumina substrates (4 × 4 mm) with interdigital Pt electrodes were carefully cleaned and placed on a hot-plate with a temperature of about 100 °C. Concentrated solution was then drop-casted onto the sensor substrates using micro-pipettes. The thickness of the thin films was about 1 µm and was controlled by the volume of the solution in the micro-pipettes. After coating, the alumina substrates were heated at 100 °C in an oven for 1 h to eliminate the remaining solvent and then sintered at 200 °C in Ar gas for 1 h to improve the adhesion and contact. Pure graphene-based sensor as the control sample was also prepared using the same method.Two as-prepared dispersions containing equivalent weights of g-C3. Nitrogen gas was used as the carrier gas. NO2 gas with a starting concentration of 500 ± 1 ppm in nitrogen was used as the target gas. The accurate concentration control of the target gas was achieved by using a mixing system equipped with mass flow controllers  and mass flow meters. All the measurements were conducted under ambient condition. The total gas flow of the carrier and target gases was kept at 250 sccm throughout the measurement process. The electrical conductance signal of the sensors was collected and recorded by data acquisition  through a customized clamp and wire connector. The sensing response was defined as:NR and GR represented the resistance of the sensors upon exposure to nitrogen and target gas, respectively.The sensors were placed in a stainless chamber having a total volume of 10 cm3N4 or g-C3N4/graphene nanocomposite onto an oxidized silicon wafer, followed by a drying process at 80 °C for 12 h. X-ray diffraction (XRD) was performed using a Rigaku RINT2200 X-ray diffractometer  with monochromatized Cu-Kα radiation. Atomic force microscopy (AFM) measurements were carried out on a Bruker MutiMode-8 system . The microstructure, morphology, and elemental distribution of the samples were systematically investigated by Raman spectroscopy , Scanning Electron Microscopy , and Energy-dispersive X-ray spectroscopy (EDS) mapping (co-equipped with SEM) , respectively. Chemical compositions and element binding energies were analyzed using X-ray photoelectron spectroscopy (XPS) on an Ulvac-phi Veresprobe II system  with monochromatic Al Kα as an excitation source. The absorption spectroscopy measurements were performed using a Varian Cary 6000i and a 1 cm cuvette.For material characterization, samples were prepared by drop-casting the solution containing g-C3N4 nanosheets from bulk g-C3N4 using a facile HCl acid treatment followed by an ultrasonic process. The effect of acid treatment on the exfoliation result was carefully examined. The exfoliated g-C3N4 nanosheets exhibited a uniform thickness of about 3–5 nm and a lateral size of about 1–2 µm. g-C3N4/graphene nanocomposite was prepared via a self-assembly process. Remarkably, the restacking phenomenon of g-C3N4 was greatly alleviated after compositing with graphene. A promising sensing performance of g-C3N4/graphene nanocomposite toward NO2 gas at room temperature was demonstrated. The nanocomposite sensor exhibited better recovery as well as two-times the response compared to the pure graphene sensor. The promising performance of the nanocomposite sensor was attributed to a synergetic effect in which the graphene with superior carrier mobility acts as the signal pathway, while the g-C3N4 nanosheet, with an active surface, plays the role of analyte acceptor.In summary, we successfully prepared g-C


The use of antibodies to target immune checkpoints, particularly PD-1/PD-L1, has made a profound impact in the field of cancer immunotherapy. Here, we identified KN035, an anti-PD-L1 nanobody that can strongly induce T-cell responses and inhibit tumor growth. The crystal structures of KN035 complexed with PD-L1 and free PD-L1, solved here at 1.7 and 2.7 Å resolution, respectively, show that KN035 competes with PD-1 (programmed death protein 1) for the same flat surface on PD-L1, mainly through a single surface loop of 21 amino acids. This loop forms two short helices and develops key hydrophobic and ionic interactions with PD-L1 residues, such as Ile54, Tyr56 and Arg113, which are also involved in PD-1 binding. The detailed mutagenesis study identified the hotspot residues of the PD-L1 surface and provides an explanation for the stronger (~1 000-fold) binding of KN035 to PD-L1 than PD-1 and its lack of binding to PD-L2. Overall, this study reveals how a single immunoglobulin-variable scaffold of KN035 or PD-1 can bind to a flat protein surface through either a single surface loop or beta-sheet strands; and provides a basis for designing new immune checkpoint blockers and generating bi-specific antibodies for combination therapy. Immune checkpoints are inhibitory pathways of the immune system that maintain self-tolerance and prevent autoimmunity . The PD-−/− mice do not express PD-L2, leading to increased airway hyperactivity and lung inflammation [de novo design of PD-1/PD-L1 inhibitors.However, these immune checkpoints are often upregulated by tumor cells to induce local immune suppression and attenuate the endogenous antitumor immune response , 17. Forammation . Therefoammation ,  21, leammation . Here, wammation , designaammation , 26. NotKd value of 3.0 nM  from pre-formed PD-1/PD-L1 complexes slightly more effectively than durvalumab   and screened the heavy-chain-only nanobody phage library derived from a PBMC  with conventional panning technology . One of f 3.0 nM . KN035-Ff 3.0 nM . KN035-Frvalumab , which iin vitro . As KN03rization  was chos dosages , which ivalumab) . These rN-terminal immunoglobulin-variable domain of PD-L1 at 1.7 Å resolution and with free PD-L1 at 2.7 Å resolution. The models were built and refined to obtain good geometry   geometry . The crygeometry . The oveys111.3) , and the2  were isolated 1 week after the last immunization. The sequence corresponding to the variable domains of the heavy-chain antibodies was amplified with specific primers and used to create a nanobody phage display library. Enrichment screening against PD-L1-Fc was performed in 96-well plates coated with 10 μg of protein per well. High-affinity bacteriophages were obtained after 4 rounds of screening. Ninety-six individual colonies were randomly selected and amplified in culture. Positive colonies verified by ELISA (enzyme-linked immunosorbent assay) were sequenced and defined. The nanobody coding genes were cloned into pET-32b  and expressed in Escherichia coli. The nanobodies were purified and screened for their ability to block the interaction between PD-L1 and PD-1 by performing a competitive ELISA. One of the nanobodies showing strong activity in blocking the PD-L1/PD-1 interaction was termed KN035. The coding DNA sequence for KN035 was fused with that of the coding fragment for human IgG1 Fc (KN035-Fc) and cloned into vector pcDNA3.1. KN035-Fc was expressed in HEK293 cells and secreted in culture medium. It was purified to homogeneity (>95%) by performing Protein A affinity chromatography .An anti-PD-L1 nanobody was produced and purified as previously described . In brie2 atmosphere for 2–3 h. After incubation, all the suspension cells were carefully removed, and the medium was changed to complete Roswell Park Memorial Institute-1640 medium with 5 ng ml−1 granulocyte-macrophage colony-stimulating factor  and 20 ng ml−1 interleukin-4. The medium was refreshed every 2 days, and 10 ng ml−1 tumor necrosis factor-α was added on the 4th day. Finally, the mature dendritic cells were harvested for mixed lymphocyte culture reaction experiments on the 5th day. CD4+ T cells (1×105) and allogeneic dendritic cells (1×104) were co-cultured with or without dose titrations of KN035-Fc or the control antibody or durvalumab [t-tests.PBMCs were obtained from heparinized peripheral blood samples of healthy donors using Ficoll–Hypaque density gradient centrifugation . Freshlyrvalumab , 48. Thein vivo, a xenograft mouse model [6 A375-PD-L1 cells (50 μl) and 1×106 human PBMCs (50 μl) [−1 and 0.037, 0.18 or 0.92 mg kg−1, respectively (same molar amount for both). Tumor volumes were measured along three orthogonal axes  and calculated as tumor volume=(abc)/2. Mice with tumor volumes larger than 2 000 mm3 were killed by CO2 administration. Statistical analyses were performed using GraphPad Prism software . The in vivo activity of KN035-Fc and durvalumab were also tested on a B16-PD-L1/C57 mouse model; however, both antibodies showed limited tumor-suppressing effect at the same dosages.To evaluate the antitumor effect of KN035-Fc se model , 47 was E. coli BL21 (DE3) as inclusion bodies. Cells were cultured at 37 °C in Lysogeny broth and induced with 0.5 mM IPTG  until the optical density at 600 nm reached 1.0. After a further 16-h incubation at 37 °C, the cells were collected by centrifugation, resuspended in lysis buffer  and lyzed by sonication. Inclusion bodies were recovered by centrifugation at 15 000 g for 10 min and were then washed three times with lysis buffer, followed by washing without Triton X-100. The inclusion bodies were dissolved in 20 mM Tris, pH 7.4, containing 6 m GuHCl, 0.5 mM ethylenediaminetetraacetic acid and 10 mM dithiothreitol. The solubilized fraction was clarified by centrifugation and dialyzed against 10 mM HCl to remove dithiothreitol. After dialysis, the sample was redissolved in 6 M GuHCl and added drop-wise into a refolding buffer consisting of 1 m Arg hydrochloride, 0.1 m Tris, pH 8.0, 2 mM ethylenediaminetetraacetic acid, 0.25 mM oxidized glutathione and 0.25 mM reduced glutathione. After incubation at 4 °C overnight, the complex was dialyzed against 10 mM Tris, pH 8.0 and purified to homogeneity using a HisTrap Ni-Sepharose column, HiTrap SP ion-exchange column and Superdex 75 . Other PD-L1 variants, such as I54A, Y56A, E58A, D61A, N63A, Q66A, V68A, R113A, M115A, S117A, Y123A and R125A, were prepared using the same procedure. For the preparation of the PD-L1/KN035 complex, the N-terminal IgV domain of PD-L1 (amino acids 19–132) was similarly cloned into pET-28a and expressed in E. coli as PD-L1. Refolding was performed in refolding buffer containing 0.1 mg ml−1 of KN035. The PD-L1 IgV domain/KN035 complexes were subsequently purified using ion-exchange and gel filtration columns .Genes encoding human PD-L1 (amino acids 19–239) were cloned into pET-28a. Proteins were expressed with a C-terminal His-tag in −1 and screened for crystallization conditions using commercially available buffer with sitting-drop vapor diffusion; 0.2 μl of the protein complex solution was mixed with 0.2 μl of reservoir solution. Diffraction-quality crystals of PD-L1-IgV/KN035 were obtained at room temperature from 1.4 m ammonium sulfate and 2 m sodium chloride after optimization. The crystals of PD-L1 were grown with a precipitation solution of 0.2 mM ammonium acetate and 20% PEG 3350.Both purified PD-L1 and PD-L1/KN035 complexes were concentrated to ~15 mg mlCrystals were cryoprotected in 20% glycerol in the mother liquor and flash-cooled in liquid nitrogen. Diffraction data were collected on beamlines BL17U1 (SSRF) and BL19U1 (NCPSS), Shanghai, China . The dat–1 KN035 for 40 s before equilibration for 60 s in phosphate-buffered saline with 1% bovine serum albumin. Variants of PD-L1 were prepared as a twofold serial dilution in 0.1% bovine serum albumin and separately incubated with the KN035 bound on the tips for 120 s. Then, the PD-L1 variants were allowed to dissociate for up to 320 seconds depending on the observed dissociation rate. All measurements were corrected for baseline drift by subtracting a control sensor exposed to running buffer only. Data analysis and curve fitting were carried out using Octet software. The affinity of these PD-L1 variants toward PD-1 could not be accurately measured because the affinity between PD-1 and PD-L1 is very low (~6–8 μM) [Kd values of 2.1 nM and 0.84 nM, respectively (data not shown). These Kd values are largely consistent with the Kd (3.0 nM) derived from KN035-Fc fixed on protein A chip, with differences likely arising from avidity and protein-immobilizing methods.A fortéBio Octet K2 instrument was used to measure the binding kinetics of PD-L1 variants to KN035-Fc with protein A sensor . All sen~6–8 μM) , 40, and


Graphene has become a bridge across optoelectronics, mechanics, and bio-chemical sensing due to its unique photoelectric characteristics. Moreover, benefiting from its two-dimensional nature, this atomically thick film with full flexibility has been widely incorporated with optical waveguides such as fibers, realizing novel photonic devices including polarizers, lasers, and sensors. Among the graphene-based optical devices, sensor is one of the most important branch, especially for gas sensing, as rapid progress has been made in both sensing structures and devices in recent years. This article presents a comprehensive and systematic overview of graphene-based microfiber gas sensors regarding many aspects including sensing principles, properties, fabrication, interrogating and implementations. Microfibers with sub-wavelength geometry and large index contrast between the fiber core and surroundings ,2 has beGraphene is a unique two-dimensional material composed of carbon in a honeycomb lattice with atomic thickness ,8, and hAmong them, graphene-based gas detection is one of the most creative and successful applications, showing the potential to achieve ultimate sensitivity: single molecule gas detection . MoreoveWhen deposited on microfibers, graphene interacts with the evanescent field, enabling different kinds of graphene-based microfiber gas sensors. Here we conclude the recent progress in this fiend regarding their sensing principles, fabrication and performances. Typical graphene-based microfiber gas sensors include biconical tapers, gratings, interferometers, coils, ring cavities, and so on. These fiber-optic graphene gas sensors are categorized in both passive (such as interferometric) and active (such as pumping laser-based) techniques, which shows the potential applications in highly-sensitive gas sensing in combining with microfiber and new materials. Although some review articles on graphene-based electrical gas sensors have been published ,10, ther±E(κ) F|κ|= ±ħν, where κ is the wave vector, Fν ≈ 106 m/s is the Fermi velocity, and ħ is Planck’s constant  + 1}−1 is the Fermi–Dirac distribution, ħ = 1.05 × 10−34 J·s, Bk = 1.3806505 × 10−23 J/K is Boltzmann’s constant, and e = −1.6 × 10−19 C is the unit charge. External electric tuning majorly influences the g,interσ, which is also directly related to the saturable absorption, which is driven by the photoexcited electron kinetics [Re(gσ) and Im(gσ) are the real and imaginary parts of gσ, respectively. By regarding the graphene monolayer thickness Δ = 0.4 nm, the refractive index of the graphene layer can be derived from [The theoretically calculated results are shown in kinetics . Consideved from :(5){Re [effn vs. |FE|” correlation of a graphene-based microfiber with core diameter ≈ 8 µm. It can be seen that by changing the Fermi level of graphene, both the transmission phase and the transmission loss of a graphene-based microfiber could be modified remarkably. Such a mechanism can not only be applied to sensing, but has also been widely used for fast optical modulation [Once incorporated with a microfiber, the graphene performs as a part of the cladding, and can influence the optical transmission via mode effective refractive index modulation. According to different graphene–microfiber hybrid geometries, the mode field distributions and complex effective index od (FEM) ,44. Figuod (FEM) ,45, loweod (FEM) . The moddulation ,48.A is the optical amplitude, Re(effn) and Im(effn) are the real and imaginary parts of the graphene-based microfiber, z is the transmission distance, t is the time delay, c is the light velocity in vacuum, and ω is the optical frequency. As a conclusion, the Fermi-level modification of graphene would modulate both the output phase and output power of the microfiber. In optical nonlinear processes, such an effect also alters the phase-matching and nonlinear threshold [It is known that the electromagnetic field distributed along microfibers obeys the Bessel equations in cylindrical coordinates , which hhreshold .3, NO2, and H2O. It is worth pointing out that for graphene oxide (GO) or other functionalized graphene, non-polar gas such as H2 is also detectable [When gas molecules are adsorbed on the surface of graphene, the Fermi level of graphene is tuned ,55. Figutectable . Figure tectable . As expoSince the first graphene-based microfiber optical gas sensor reported in 2012 , a varieTypically, microfibers are fabricated from commercial glass fibers or bulks, by using the fusing & drawing method , which cSometimes one not only wants a microfiber-guiding evanescent field, but also hopes to keep the inner microstructures of the original fiber ; then, chemical etching becomes a choice ,68. For 4 gas in a dilute H2 environment over the surface at 1000 °C. With the exposure of Cu foil in a CH4/H2 environment, the nucleation of graphene islands starts taking place randomly, eventually aggregating into a continuous graphene film. The chemical vapor deposition (CVD) and wet transfer techniques provide inch-level large-scale monolayer graphene for incorporation with microfibers ,70,71,723 solution; covering the PMMA/graphene flexible film on a substrate or wrapping it on a microfiber; removing the PMMA by using acetone, leaving only graphene remaining.Graphene can be either attached on or wrapped around a microfiber, as schematically shown in To characterize the quality of graphene on the microfibers, Raman spectroscopy ,78,79, sIn recent years, graphene gas sensors with microfibers develop rapidly, some of them are also reviewed in Refs. ,82,83,84Based on the graphene-attached microfiber scheme, we reported gas sensors based on both optical intensity detection and interferometric demodulation by using mode field analysis or Mach-Zehnder Interferometer (MZI) ,84. Figu3 gas detection [3 trace detection, 0.3 ppm resolution and 0.5 s response delay was achieved.The regeneration time for the acetone gas detection was in the rage of several minutes. The interferometer scheme was a big step forward to achieve a much higher sensitivity. etection . It illu2 gas. A sensitivity of 0.5 ppm was achieved in this work. As the reduced GO film was thicker and fluffier than the CVD graphene, its response time was limited to minute-level. In this type of sensor, it is also important to enhance the mode distribution ratio out of the core and optimize the microfiber diameter (or the thickness of the remained cladding), as discussed by Zhang et al. in Ref. [H ~ ln(S)ln(D)/A, the best diameter could be around 10 µm for graphene-wrapped silica fiber-Bragg-gratings (FBGs) with standard 8 µm core.Similarly,  in Ref. . Figure 3 gas sensing and NO2 gas sensing, respectively. As shown in pλ = 2effΛn, where Λ is the MFBG period. The gas adsorption based on effn modulation was measured by detecting the spectral shift of the MFBG. The maximum sensitivity of the CVD graphene-coated MFBG reached 0.2 ppm for NH3 gas and 0.5 ppm for xylene gas, respectively.Since the scheme of graphene-microfiber attachment spatially limits the light–graphene interaction, since 2014, graphene-wrapped microfiber structures have become a trend. For instance, based on graphene-wrapped micro fiber Bragg gratings (MFBGs), the footprint of the sensors can be dramatically miniaturized. Wu et al.  and Srid21 mode is more sensitive to local refractive index alteration than the in-core HE11 [3 gas detection and ~0.2 ppm for H2O vapor detection were achieved. By using GO-ZnO film, Hu et al. also recently realized a NH3 gas sensor, demonstrating sub-ppm sensitivity, as shown in 3 gas. Additionally, in 2014, Mishra et al. also reported a graphene Surface Plasma Resonance (SPR) sensor based on microfibers for sensing NH3 gas [Another method to enhance light–graphene interaction in graphene-based microfiber structures is to excite high-order mode propagation with larger mode-field area or to excite plasmons. In 2014, Yao et al. demonstrated a graphene-based microfiber multimode interferometer, as shown in ore HE11 , hence i NH3 gas . Mishra Q resonance, the interferometric resolution can be effectively improved. Yu et al. made such an attempt by building a GO-deposited microfiber knot resonator [Q factor. In future investigations, by optimizing the graphene coverage region [Limited by the spectral resolution of the optical interference and the linear loss of the passive devices, sub-ppm seemed to be the limitation for a graphene-based microfiber gas sensor. Taking advantage of high esonator . Figure e region , the res3 gas detection, which is a globally leading number comparable to other advanced techniques [Av is the acoustic velocity, c is the light velocity in vacuum, pn and sn are the effective indexes of the pump mode and the generated Stokes mode, pf and sf are the pump frequency and the Stokes frequency, eγ is the electro-strictive coefficient, and BΓ is the lifespan of the phonons. Gas adsorption on the reduced GO film enables an RF spectral shift 200 kHz/ppm, while the uncertainty of the Brillouin optomechanical generation is only 200 Hz, due to the extremely high Q factor (106). Moreover, such an optomechanical micro-resonator kept an exceptional dynamic range from 1 ppb to 400 ppm, crossing over five orders.Moreover, in 2017, based on graphene-enhanced Brillouin scattering, a microfiber interrogated Whispering Gallery Mode ptomechanical gas sensor was realized. chniques ,96. SuchThe above review summarizes the progress in optical graphene gas sensors based on microfibers. By optimizing the optical sensing structures, this type of sensor has made significant advancements in sensitivity, which is summarized in 3, H2O, or NO2, and these sensors lack selectivity. Towards the realization of graphene-based microfiber sensors for other gas detection, the graphene material itself would be further functionalized, such as by using element-doped films [In this article, we review the principles, fabrications, implementations, and performances of optical graphene gas sensors with microfibers, which have attracted intense interest in research and development and play an important role in industry. The mode of “graphene and microfiber” has become a widely used platform for—but not limited to—gas detections. In this way, higher sensitivity and better selectivity are constant pursuits, while still remaining challenges. On one hand, to increase the sensitivity, more and more new optical mechanisms and techniques are being reported, such as graphene-based laser sensing , high-ored films , graphened films , or fluoed films . With th


TBX5 3′ untranslated region (UTR) variations with congenital cardiac malformations. This study aimed to investigate the relationship between TBX5 3′UTR variants and risk for congenital heart disease (CHD) susceptibility in two Han Chinese populations, and to reveal its molecular mechanism. The relationship between TBX5 3′UTR variants and CHD susceptibility was examined in 1 177 CHD patients and 990 healthy controls in two independent case–control studies. Variant rs6489956 C>T was found to be associated with increased CHD susceptibility in both cohorts. The combined CHD risk for the CT and TT genotype carriers was 1.83 times higher than that of CC genotype, while the risk for CT or TT genotype was 1.94 times and 2.31 times higher than that of CC carriers, respectively. Quantitative real-time PCR and western blot analysis showed that T allele carriers exhibited reduced TBX5 mRNA and protein levels in CHDs tissues. Compared with C allele, T allele showed increased binding affinity to miR-9 and miR-30a in both luciferase assays and surface plasmon resonance analysis. Functional analysis confirmed that miR-9 and miR-30a downregulated TBX5 expression at the transcriptional and translational levels, respectively. The assays in zebrafish model were in support of the interaction of miR-9/30a and TBX5 3′UTR . We concluded that TBX5 3′UTR variant rs6489956 increased susceptibility of CHD in the Han Chinese population because it changes the binding affinity of two target miRNAs that specifically mediate TBX5 expression.TBX5 is a vital transcription factor involved in cardiac development in a dosage-dependent manner. But little is known about the potential association of  Congenital heart diseases (CHDs) are the most prevalent type of human structural birth defects worldwide, affecting between 19 and 75 per thousand live births ,3,4,5. HTBX5 invariably result in Holt-Oram syndrome (HOS), an inherited disease characterized by upper limb and cardiac deformities [TBX5 might be associated with increased CHD risk through altered gene expression or dosage effects.Genome-wide association studies (GWASs) suggest that a large number of non-coding variants contribute to the risk of various diseases, including cardiovascular disease ,8,9,10.ormities , 17. ThuTBX5 is a transcription factor with well-defined roles in heart and forelimb development. Multiple studies in animal models have confirmed that cardiac development is sensitive to Tbx5 dosage , 18, 19.TBX5 in the promoter [TBX5 3′UTR to CHDs remains unknown. Our previous study demonstrated that miR-10a and miR-10b significantly repressed TBX5 protein levels by targeting its 3′UTR [TBX5 3′UTR that significantly increases CHD risk in a Han Chinese cohort composed of 1 177 CHD patients and 990 healthy controls. We further investigated the molecular mechanism underlying that TBX5 3′UTR variants influence risk for CHD susceptibility.Recently, several non-coding variants of promoter , intron promoter  and enhapromoter  were repts 3′UTR . In presTBX5 3′UTR were identified. Among them, four variants had minor allele frequency (MAF) >0.05 and were chosen for genotyping in 288 cases and controls of the Shandong group. Among them, the prevalence of variant rs6489956 (c.* 1101C>T) was significantly different between the CHDs and the control subjects. This variant was then selected for further validation in the other two independent case–control cohorts , as well as 272 CHDs and 384 controls in the Shanghai cohort . The combination of these two independent studies showed a 1.83-fold increased CHD risk when the dominant model is used for TBX5 rs6489956 (P=3.62×10−8) in a total number of 1 177 CHDs patients and 990 controls (P=8.21×10−8) and 2.31  for the CT and TT genotypes, respectively. Therefore, The variant rs6489956 was significantly associated with an increased risk of CHD in two cohorts separately and together. Similarly, the allele analysis showed that carriers of the susceptible T allele exhibited a higher risk of CHD than carriers of the C allele in both cohorts, resulting in a 1.86-fold combined higher susceptibility of CHD . All genotype frequencies were in accordance with the Hardy-Weinberg expectation among the control subjects (P>0.05).As the homozygous TT genotype was very rare, we applied the dominant model of inheritance to enhance the statistical power of the study, which combined homozygous TT alleles with heterozygous CT alleles to compare with the wild-type CC alleles in the association studies. Logistic regression analyses demonstrated heterozygote CT and homozygote TT subjects had a significantly increased risk of CHD compared with those with the wild-type CC allele in a study of 905 CHDs and 606 controls in the Shandong group (odds ratio (OR)=2.33, 95% confidence interval (CI)=1.70–3.19, controls , with anTBX5 c.*1101C>T (rs6489956) is related to multiple types of CHDs (P=9.70×10−6). In isolated phenotypes, similar statistical significance was noted in atrial septal defect (ASD) cases (P=3.67×10−3) and ventricular septal defect (VSD) cases (P=2.69×10−5).A stratified analysis was performed according to standard CHDs classifications as previously described , 24. It  of CHDs . NotablyTBX5 c.*1101C>T (rs648995) variant is located in the TBX5 3′UTR, we assumed that this variant might be involved in either TBX5 transcription or translation. Thus, we examined TBX5 mRNA expression using quantitative RT-PCR on the cardiac tissue samples. Of the 30 cardiac samples tested, 25 had the rs6489956 CC genotype, four had the CT genotype, and only one had the TT genotype. The relative TBX5 mRNA expression level in the CT/TT genotypes was only 49.3% of that of the CC genotype (P<0.05) (P=0.0037)  . Consist=0.0037) . AlthougTBX5 mRNA expression associated with the variant rs6489956 was further confirmed by in vitro luciferase assays in different cell lines. Consistent with the tissue sample results, the plasmid containing the mutant-T allele showed a 26%, 28% and 31% reduction in luciferase activity when compared with the wild-type C allele in HEK 293T, HCM and H9C2 cells, respectively (P<0.01)  . TherefoTBX5 3′UTR variant rs6489956, there are 16 software-predicted miRNAs including five upstream miRNAs, seven downstream miRNAs and four on-site miRNAs   . For the×10−6m) , it mighescribed .TBX5 caused by the 3′ UTR variant c.*1101C>T was demonstrated by a psiCHECK luciferase reporter assay as well as the rescue experiments from miRNA inhibitors. The luciferase gene expression of psiCHECK-C and psiCHECK-T were both inhibited in a linear manner with increased miRNAs concentration, and the miRNAs displayed a more pronounced inhibition effect on psiCHECK-T : wild-type (WT) control, standard MO control (5 ng), tbx5 MO (5 ng), tbx5 MO (2.5 ng), tbx5 MO (0.5 ng), MO (5 ng)+C allele mRNA (100 pg), MO (5 ng)+T allele mRNA (100 pg), MO (5 ng)+C allele mRNA (100 pg)+hsa-mi9 (100 pg), MO (5 ng)+T allele mRNA (100 pg)+hsa-miR-9 (100 pg), MO (5 ng)+C allele mRNA (100 pg)+hsa-miR-30a (100 pg) and MO (5 ng)+T allele mRNA (100 pg)+hsa-miR-30a (100 pg). As previously reported [tbx5 loss-of-function in zebrafish caused heart developmental defects, including different degrees of heart looping arrested, heart edematous, string-like heart, and so on, which were confirmed in our results  was found to significantly increase CHD risk in Han Chinese by interacting with miR-30a and miR-9 . Although we first postulated that a functional variant in the TBX5 3′UTR may increase the risk of CHD, a recent study indirectly supported our findings by reporting that a TBX5 enhancer variant causes isolated congenital heart disease [TBX5 3′UTR and the risk of CHD need further exploration in other ethnic groups. However, our study elucidated that the sporadic CHDs could be attributed to the combined contribution of the common regulatory variations in the TBX5 gene, as well as the epigenetic factors such as miR-30a or miR-9.It is well known that the mutations in syndrome ,34,35,365 dosage , 37, 38. disease . BecauseTBX5 non-coding variants with increased CHD risk may be explained by the sensitive nature of the dosage effect of TBX5 on heart development. While severe loss-of-function mutations in TBX5 coding regions led to both heart and forelimb phenotypes, non-coding variants were associated with compartmentalized phenotypes, which might reflect the differing sensitivities of the impacted tissues to the altered TBX5 expression level. This contention is supported by the fact that the TBX5 intronic variant rs7312625 was reported to be associated with altered PR intervals . In African Americans, and intronic variant rs3825214 was shown to reduce lone atrial fibrillation risk in Han Chinese [The possible mechanism of underlying the association of  Chinese , 40. TheTBX5 expression can be observed throughout the epicardium and myocardium, including the free walls and septa of all four cardiac chambers, while its expression in the endocardium is limited to the left ventricle [Tbx5 mRNA expression is restricted to the atria and left ventricle. Consistent with these expression patterns, HOS patients with TBX5 mutations may have a number of significant heart defects, such as septal defects and certain complex malformations , APVR and hypoplastic left heart) [n=830, P=9.70×10−6). Additionally, we speculated that the failure to detect any correlation between variant rs6489956 and APVR and outflow tract malformation susceptibility might be due to the limited and insufficient sample size.In both embryonic and adult human heart tissues, entricle . Howeverentricle  and chicentricle , Tbx5 mRt heart) , 44. In in vivo and in vitro assays on the function of variant rs6489956 indicated that the T allele led to reduction in TBX5 gene expression. We further identified two miRNAs, miR-9 and miR-30a, that interacted with this variant. This is not surprising, because miRNAs are predicted to function in the post-transcriptional regulation of nearly one third of all human genes [TBX5 mRNA, but reduced its protein concentration, while miR-9 downregulated both transcription and translation of TBX5. It has been extensively reported that miRNAs help to regulate cardiac growth and remodeling, and contribute to specific myocyte properties. miRNAs are implicated in numerous cardiovascular diseases, such as heart hypertrophy, heart failure, atherosclerosis, acute myocardial infarction, VSD, TOF and arrhythmias [TBX5 was disturbed during a sensitive time window of development, it increases the likelihood of inducing CHD. As a new model trigger, the interactions between miRNAs and TBX5 regulatory variant increase the CHD risk through the downregulated TBX5, either at the post-transcriptional or translational level.Both an genes . It was hythmias . Thus, wHand2-deficient mice [Because miRNAs are vital factors that mainly target 3′UTR sequences of mRNA, we suspected that miRNAs might have important roles in CHDs. Both bioinformatic predictions and functional analyses showed that both miR-30a and miR-9 have a higher affinity to the minor T allele. We further confirmed these results using luciferase assays, quantitative RT-PCR, western blot analysis and whole-embryo microRNA sensor assay in zebrafish. Recently, many studies have implicated miRNAs in various cardiovascular diseases. For example, miRNA-1–2 knockout mice have decreased levels of the cardiac transcription factor Hand2, which results in heart malformations (VSD) similar to those observed in ent mice , 54. ComTBX5 3′UTR variant rs6489956 on the increased susceptibility of CHD in two independent Han Chinese populations. It was well known that both Tbx5 overexpression and insufficiency result in heart defects in developing embryos. In view of the decreased TBX5 mRNA levels detected in the heart tissues of CHD patients with the CT and TT genotypes, the individuals with the T allele are susceptible to develop CHD due to the TXB5 deficiency. Functional studies clearly showed that the consequent repression of TBX5 expression on the post-transcriptional level resulted from an enhanced binding affinity of the T allele to miRNAs. Our data highlighted an important function of the underlying genetic variations in the 3′UTR of TBX5 in CHDs, providing new insight into risk assessment for these common birth defects.In summary, our study demonstrated a significant effect of We analyzed samples from two independent case–control groups. The Shandong group consisted of 905 CHD patients and 606 matched controls recruited from the Cardiovascular Disease Institute, General Hospital of Jinan Military Command  between August 2009 and September 2011. The Shanghai group was composed of 272 CHD cases and 384 matched controls enrolled in the Children’s Hospital of Fudan University  between August 2010 and November 2011. All subjects were unrelated ethnic Han Chinese. All of the controls were non-CHD outpatients from the same geographical area who were matched to the affected individuals in terms of age and sex over the same period. CHDs patients who were considered to be syndromic and not isolated CHDs, or who had a positive family history of CHD in a first-degree relative , were excluded from the study.There were no newborn samples in our study. We classified the 1 177 CHDs cases into seven broad categories according to the commonly accepted criteria . SpecifiTBX5 gene, we selected a subset of 32 subjects for resequencing. Half of these subjects were CHD patients, and half were controls. Fifty-three cardiovascular tissue samples were obtained with informed consent from CHD patients who had undergone cardiac operations from January 2011 to September 2011 in the Cardiovascular Disease Institute, General Hospital of Jinan Military Command  and Children’s Hospital of Fudan University , among which 30 were used for quantitative real-time PCR (RT-PCR) assay and 23 for western blot analysis. The 53 collected cardiac tissue samples comprised 10 atrium samples, 4 ventricle samples, 4 appendage sample, 8 atrial septum samples, 4 ventricular septum samples, 15 outlet samples, and 8 aorta samples. Normal heart tissue samples were collected by the Department of Forensic Medicine, Fudan University from adults with no history of heart disease who died of accidents.To screen for genetic variants in the 3′UTR of All study protocols were reviewed and approved by the medical ethics committee of Children’s Hospital of Fudan University, and written consent was obtained from the parents of subjects and/or the subjects themselves before study enrollment.TBX5 3′UTR fragments were amplified using PCR to amplify a 1757-bp fragment from 114793401 to 114791645 (NM_000192) in 32 unrelated individuals randomly selected for variant screening by sequencing. Direct dye terminator sequencing of the PCR products was carried out using the ABI Prism BigDye system according to the manufacturer’s instructions . Selected single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) >0.05 were genotyped using SNaPshot analysis (ABI). In the first stage of the present study, a total of 288 CHD cases and the same number of controls of the Shandong group were collected and genotyped in October 2010. The variants associated with CHD risk were chosen for further case–control study in the following Shandong and the Shanghai samples. The samples were sequenced and genotyped on an ABI 3730 automated sequencer and analyzed using Lasergene 7.0 and Peakscanner 1.0, respectively. All of the PCR and sequencing primers were listed in Genomic DNA was isolated from the peripheral venous blood using conventional regents. Total RNA was extracted from human cardiovascular tissue samples preserved in RNAlater  and converted to cDNA using random hexamers, oligo(dT) primers and PrimeScript RT reagent Kit . Quantitative Real-time PCR was performed with SYBR Premix Ex Taq (TaKaRa) using a StepOne Real-Time PCR system (ABI) with β-actin as an internal reference gene. Each reaction was performed in triplicate. The primers were described in Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide electrophoresis gels and blotted on to polyvinylidene fluoride membranes . Non-specific binding was blocked with 5% skimmed milk in TBS. Blots were probed with either mouse horseradish peroxidase (HRP)-conjugated anti-β-actin monoclonal antibody , or rabbit anti-TBX5 polyclonal antibody  followed by HRP-conjugated goat anti-rabbit secondary antibody . Proteins were detected using ECL reagents (Millipore).TBX5 3′UTR containing either the C or T allele was amplified from genomic DNA and subcloned into the downstream region of Renilla luciferase using XhoI and NotI restriction sites in psiCHECK-2 vector . Primers were listed in To construct the reporter plasmids, TBX5 ORF fused to PCMV6-XL4 was purchased from Origene Technologies . The mutant TBX5 was generated by the the Quick Change Site-Directed Mutagenesis Kit (Stratagene). Constructs were linearized using PvuII and the capped and polyA tailed mRNA was synthesized with mMessage mMACHINE T7 Ultra Kit  for the zebrafish microinjection.Human wild-type Human embryonic kidney 293T (HEK 293T), human cardiac myocytes (HCM) and rat cardiac myocyte (H9C2) cells were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum.5, 2.0×105 and 1.0×105 per liter, respectively. Twenty-four hours later, the cells were transfected with 400 ng of each TBX5 reporter plasmid using Lipofectamine 2000 . To screen the predicted candidate miRNAs, HEK 293T cells were co-transfected with 25 ng of psiCHECK-C/T plasmid and 75 ng of miRNA expression vector in 96-well culture plates. To further explore the interaction between TBX5 3′UTR and selected candidate miRNAs, HEK 293T, HCM or H9C2 cells were co-transfected with 50 ng psiCHECK-C/T plasmid as well as 0 ng, 50 ng, 100 ng, 150 ng or 200 ng of miRNA expression vector. For small interfering RNA assays, HEK 293T cells were co-transfected with 100 ng psiCHECK-C/T plasmid and 100 nm miRNA inhibitors  in 24-well culture plates.HEK 293T, HCM or H9C2 cells were seeded in 24-well culture plates at the density of 4.0×10Cells were lysed 24 h after transfection for luciferase assays that employed the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Three independent transfection experiments were performed, and each luciferase assay was conducted in triplicate.m of miRNA inhibitors in 6-well culture plates. Cells were harvested and lysed 48 h after transfection in RIPA buffer and assayed for the relative protein concentrations of intracellular TBX5  and β-actin (Kangchen Bio-tech) using western blot analysis. RNA was also purified from these cells for quantitative RT-PCR analysis of TBX5 mRNA levels.HEK 293T cells were transfected with 2 μg of miRNA expression vector or 100 nm. Single-stranded RNA  harboring 32 bp of 3′UTR wild-type-C allele RNA or 3′UTR mutant-T-allele was diluted into a variety of concentrations in PBST buffer  and flowed over the biotinylated miRNAs. At the end of each cycle, 5 mm NaOH was applied to regenerate the sensor surface. Data were converted by BIA evaluation software.SPR analysis was conducted using the ProteOn XPR36 protein interaction array system . Biotinylated miRNAs were immobilized to an individual channel of the streptavidin-modified sensor chip at a fixed concentration of 400 nTBX5 mRNA and 100 pg miRNAs using a microinjector . Twelve hours post-injection, the dead embryos were removed, leaving only viable embryos that were used for further analysis. Consistent with the previously published studies [−1 tricaine/1% 1- phenyl-2-thiourea  and embedded in 0.6% low melting agarose. Confocal imaging analysis was performed using Imaris software. Two transgenic zebrafish lines: Tg(vmhc:eGFP) and Tg(vmhc:mCherry-NTR) were used as described in previous work [The morpholino (MO) antisense oligonucleotide MO-Tbx5a (5′-GAAAGGTGTCTTCACTGTCCGCCAT-3′) was purchased from Gene Tools . Human miR-30a, mir-9 and a negative control were purchased from Thermo Fisher Scientific . Zebrafish were raised under standard conditions at 28.5 °C. Each 1-2-cell stage embryo was injected with a constant injection of 5 ng MO, 100 pg  studies , 55, allous work .χ2-test. Differences in demographic features and allelic or genotypic frequencies between the CHD cases and controls were evaluated using Pearson’s χ2-test. To evaluate associations between the genotypes and CHD risk, ORs and 95% CIs were calculated by unconditional logistic regression analysis with adjustments for age and sex. In multiple comparison testing, we employed the Bonferroni correction. The significance level was adjusted to P=0.0025 given the four SNPs being tested for 5 genetic models in 288 cases and the same number of controls.The Hardy-Weinberg equilibrium test was performed on controls using the t-test with Bonferroni correction. The P<0.05 was defined as statistical significance. Statistical analyses were conducted with SPSS 16.0 software .Data were calculated as the mean±s.d. Significant differences between the test and control groups were analyzed by means of an independent


It is reported recently Tropomyosin-related receptor Kinase B (TrkB) plays key roles in the anoikis resistance during the processes of tumorigenesis and metastasis. However, its prognostic significance for clinical patients remains inconclusive. In order to establish a correct and practicable link between increased TrkB and prognostication of human solid tumors, a meta-analysis was performed in this article. A systematic literature research in the electronic databases PubMed, Embase and Web of Science was performed to identify eligible studies. A fixed-effects meta-analytical model was employed to correlate TrkB expression with OS, DFS and clinicopathological features. A total of 11 studies covering 1516 patients with various solid tumors were recruited in this meta-analysis. TrkB over-expression was associated with poorer OS and poorer DFS in multivariate analysis. Additionally, the pooled odds ratios (ORs) indicated that TrkB over-expression was associated with large tumor size, lymph node metastasis, distant metastasis and a higher clinical stage. Overall, these results indicated that TrkB over-expression in patients with solid tumors might be related to poor prognosis and serve as a potential predictive marker of poor clinicopathological prognosis factor. Anoikis is a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix –3. AnoikTropomyosin-related receptor Kinase B (TrkB) is a tyrosine kinase receptor for brain-derived neutrophic factor (BDNF), which triggers several intracellular signals –10. TrkBIn this study, we conducted a meta-analysis to investigate the significance of over-expressed TrkB in the prediction of prognosis of corresponding patients. The results showed that increased TrkB expression in patients with solid tumors related to poor prognosis and served as a potential prediction of poor clinicopathological prognosis factor.The initial search identified 315 publications, of which, 18 studies were of acceptable relevance. However, four of these studies were excluded because the survival curve was based on NTRK2 expression, and three were excluded because of the absence of information about clinicopathological characteristics. Ultimately, 11 studies met the eligibility criteria and were included in the current meta-analysis  was observed when using a fixed-effects model to analyze the pooled HR of the OSs.The pooled hazard ratio (HR) revealed that over-expressed TrkB was significantly associated with poor overall survival (OS) for cancer victims in multivariate analysis .A significant correlation between over-expressed TrkB and disease-free survival (DFS) was also observed in the patients with solid tumors in multivariate analysis , which triggers several intracellular signals. TrkB is over-expressed in various human malignancies and growing evidence demonstrated its association with tumour cell proliferation, invasion and metastasis –31. TrkBIn this meta-analysis, we evaluated survival data from 1516 solid tumor patients from 11 eligible studies, which met the inclusion criteria, were organized according to OS and DFS. For all studies, TrkB expression was detected by IHC. By meta-analysis of the 11 studies, we identified the pool HRs which indicated that TrkB was a factor in poor prognosis in various cancers. Because there is no significant heterogeneity among our included studies, so we did not perform further subgroup analyses. Additionally, no publication bias was observed. These results indicated over-expressed TrkB was positive related to poor OS and DFS in solid tumor patients. In addition, pooled results of the correlations were identified between over-expressed TrkB and clinicopathological features of patients with solid tumors. The results showed the expression of TrkB was positively associated with large tumor size, lymph node metastasis, distant metastasis and a higher clinical stage. We can explain this result by TrkB’s ability to enhance anoikis resistance, promote tumour cell proliferation, invasion and metastasis. Because of its involvement in these processes, TrkB is likely to be causally involved in tumor progression and, consequently, increased levels of TrkB would be expected to indicate a poor prognosis.This meta-analysis was properly conducted, however, further analysis with several limitations would be considered in the future. first, need more trials to analysis; second, some of the survival data were extracted from Kaplan-Meier curves and might be less reliable than a direct analysis of variance; third, we need to search more non-English publications. In addition, the possible existence of unpublished studies could also result in potential publication bias. In general, concerning these limitations mentioned above, we must interpret these results with adequate caution.In conclusion, our meta-analysis indicated that over-expressed TrkB was positive related to poor OS and DFS in solid tumor patients. Over-expressed TrkB could be served as a potential marker for poor clinicopathological prognostic factors in patients with various solid tumors. However, additional studies related to specific tumor types are necessary to illuminate the clinical utility of increased TrkB in solid tumors.This systematic review and meta-analysis is reported in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement. The literatures relevant to TrkB expression and survival in solid tumors were searched in the PubMed, Embase and Web of Science databases through March 31, 2017. The search terms included the following key words in various combinations: TrkB, NTRK2, prognosis, prognostic, survival, and overall survival. The hits were limited to human studies and those published in English. The references list of review and bibliographies were further sifted to identify additional potentially relevant studies to avoid omission due to the electronic search approach.The collected studies included in this meta-analysis had to meet the following criteria: (1) a pathological diagnosis of cancer was made; (2) TrkB expression in patients with any type of tumor was measured via immunohistochemistry; (3) associations of TrkB expression with OS, DFS or clinicopathological features were described; (4) HRs and 95% confidence intervals (CIs) were reported or could be calculated (based on the information in the paper); and (5) when the same author reported repeated results from the same population, the most complete report was included. The exclusion criteria for this meta-analysis were as follows: (1) unpublished papers; (2) laboratory articles, reviews and letters; (3) non-English language articles; (4) overlapping articles or ones with duplicate data; (5) articles with only animal experiments; (6) studies without information about survival curves; and (7) TrkB expression in patients with any type of tumor was analyzed only using RT-PCR method.All data were extracted independently by two investigators (Chunze Zhang and Dan Gao). For each eligible study, the following characteristics were extracted: first author’s name, publication year, region, type of cancer, number of patients, patients’ ages, follow-up times, detection methods, staining position, cut-off values, survival data (including OS and DFS) and clinicopathological parameters, such as tumor differentiation, tumor size, lymph node metastasis and TNM stage. For studies that presented only Kaplan-Meier curves was used to extract the survival data. The Newcastle-Ottawa-Scale (NOS) was adopted to assess the study quality of each individual study. The NOS score ranged from 0 to 9, and studies with NOS score ≥ 7 were defined as high-quality studies.Q test) and I-squared statistical test were used to analyze the heterogeneity between studies. When the result of a Q-test (I2 > 50% or P < 0.05) indicated heterogeneity, the random-effects model was used for the meta-analysis. Otherwise, a fixed-effects model was used. HR with its value over 1.0 indicated poor prognosis patients with increased TrkB expression. Funnel plots were used to graphically represent the publication bias. Begg’s (rank correlation) test was adopted to confirm the publication bias.This meta-analysis was performed using Stata 12.0  software. Pooled estimates of HRs and their 95% CIs were used to estimate the association between TrkB expression and patients’ survival. The chisquared test (Cochrane’ s