TDP-43 has been identified as a disease-associated protein in several chronic neurodegenerative disorders and increasing evidence suggests its potentially pathogenic role following brain injuries. Normally expressed in nucleus, under pathological conditions TDP-43 forms cytoplasmic ubiquitinated inclusions in which it is abnormally phosphorylated and cleaved to generate a 35 and a 25 kDa C-terminal fragments. In the present study, we investigated age-related expression patterns of TDP-43 in neurons and glia and its role as modulator of inflammation following ischemic injury.Wild-type and TDP-43 transgenic mice of different age groups were subjected to transient middle cerebral artery occlusion. The role of TDP-43 in modulation of inflammation was assessed using immunofluorescence, Western blot analysis, and in vivo bioluminescence imaging. Finally, post-mortem stroke human brain sections were analyzed for TDP-43 protein by immunohistochemistry.We report here an age-related increase and formation of ubiquitinated TDP-43 cytoplasmic inclusions after stroke. The observed deregulation in TDP-43 expression patterns was associated with an increase in microglial activation and innate immune signaling as revealed by in vivo bioluminescence imaging and immunofluorescence analysis. The presence of ubiquitinated TDP-43 aggregates and its cleaved TDP-35 and TDP-25 fragments was markedly increased in older, 12-month-old mice leading to larger infarctions and a significant increase in in neuronal death. Importantly, unlike the hallmark neuropathological features associated with chronic neurodegenerative disorders, the TDP-43-positive cytoplasmic inclusions detected after stroke were not phosphorylated. Next, we showed that an increase and/or overexpression of the cytoplasmic TDP-43 drives the pathogenic NF-κB response and further increases levels of pro-inflammatory markers and ischemic injury after stroke in age-dependent manner. Finally, analyses of the post-mortem stroke brain tissues revealed the presence of the cytoplasmic TDP-43 immunoreactive structures after human stroke.Together, our findings suggest that the level of cytoplasmic TDP-43 increases with aging and may act as an age-related mediator of inflammation and neuronal injury after stroke. Thus, targeting cytoplasmic TDP-43 may have a therapeutic potential after stroke.The online version of this article (10.1186/s12974-018-1350-y) contains supplementary material, which is available to authorized users. Stroke is a leading cause of death and the major cause of long-lasting disabilities in industrialized countries . IndeedIn a search for proteins that may affect microglia/neuron immune crosstalk in aging brain, we focused our study on transactive response (TAR) DNA binding protein 43 (TDP-43). Generally localized in the nucleus, TDP-43 belongs to the family of heterogeneous nuclear ribonuclear proteins that are highly conserved in different species . TDP-43 The current study was designed to identify and characterize the age-related expression patterns of TDP-43 in neurons and microglia and to evaluate its role as modulator of inflammation following ischemic injury. We report here an age-related increase and long-lasting mislocalization of TDP-43 after stroke. The observed accumulation of cytoplasmic TDP-43 was associated with an increase in microglial activation and innate immune signaling seen by in vivo bioluminescence imaging and immunofluorescence analysis. The presence of ubiquitinated TDP-43 aggregates and its cleaved TDP-35 and TDP-25 fragments was markedly increased in older, 12-month-old mice, which showed larger infarctions alongside with an increase in neuronal death. We next showed that increase and/or overexpression of the cytoplasmic TDP-43 drives the NF-κB response and further increase levels of pro-inflammatory markers and ischemic injury after stroke. Overall, our results suggest that TDP-43 may act as an age-related modulator of inflammation after stroke. Based on our results, we propose that therapies targeting cytoplasmic TDP-43 may have a potential to modulate post-ischemic inflammation and to protect dying neurons in the ischemic microenvironment. Of note, the post-mortem analysis of the brains autopsied at different time points after human stroke suggests the presence of TDP-43 immunoreactive structures localized in the cytoplasm of the neurons in periphery and the core region of the ischemic lesion.Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care.The wild-type (C57Bl/6) mice of 3 and 12 month old (representing a middle aged mouse group) were selected for study. The TLR2-luc-GFP transgenic reporter mice were developed, validated, and genotyped as described previously . These aTransient focal cerebral ischemia was induced by unilateral left middle cerebral artery occlusion (MCAO) as described . Wild-tyd-luciferin (150 mg/kg bw), a substrate for luciferase dissolved in 0.9% saline. The mice were then anesthetized in 2% isoflurane in 100% oxygen at a flow rate of 2 L/min, placed in a heated light-tight imaging chamber. All the animals were imaged before for baseline expression and then continued at different time points post MCAO. Images were captured using a high sensitivity CCD camera with wavelengths ranging from 300 to 600 nm and exposure time for imaging of brain was set for 1 min. The bioluminescence emission was quantified by determining the total number of photons emitted per second (p/s) using live image 2.5 acquisitions and imaging software. Region of interest measurements were used to convert surface radiance (p/s/cm2/sr) to source flux or the total flux of photons expressed in photons/second. The data are represented as pseudo-color images indicating light intensity , which were superimposed over gray-scale reference photographs [The images were obtained by using IVIS 200 imaging system . Twenty minutes prior to imaging session, the mice were administered with tographs .2PO4, 5 mM MgCl2, phosphatase, and protease inhibitors), and homogenized by applying two strokes in a glass homogenizer. The suspension was incubated for 10 min on ice and then homogenized by applying six strokes in a motorized homogenizer at 250 rpm. Differential centrifugation was performed after restoration with 100 μl of 2.5 M sucrose. The first round of centrifugation was performed at 6300×g for 10 min at 4 °C. The pellet was collected and suspended in TSE buffer  and homogenized with 30 strokes using a motorized Teflon potter at 250 rpm. The obtained suspension was centrifuged at 4000×g for 5 min. The resulting supernatant was discarded, and the pellet was washed with TSE buffer until the supernatant was clear. Pellet was then suspended in RIPA buffer  with 2% SDS as nuclear fraction. The supernatant collected from the first round of differential centrifugation was subjected for centrifugation at 10,700 g for around 30 min and the supernatant collected was used as cytoplasmic fraction. The protein lysates from different fractions were quantified by Bradford and subjected for Western blot and co-immunoprecipitation as described previously [Cytoplasmic and nuclear fractions were obtained as per the protocol described earlier , 27. Fiveviously .The mice were anesthetized and perfused transcardially with phosphate buffer solution followed by 4% paraformaldehyde at pH 7.4. The brain tissue was post fixed overnight in 4% paraformaldehyde and then cryo-preserved in 30% sucrose. The following procedure was adopted for the study as described earlier . The fixThe mice were anesthetized and perfused transcardially using phosphate buffer saline followed by 4% paraformaldehyde solution (pH 7.4). The brains were then sectioned to 25-μm-thick slices and stained with cresyl violet histological stain. The mean stroke area of approximately ten sections from 3 to 12 months old were calculated after 72 h post MCAO by using ImageJ software and expressed as % stroke area.  × 100) .The mouse cytokine array  was used to detect the levels of different cytokines in sham, acute, chronic stroke-operated mice. The array was performed according to the manufacturer’s instructions. The protein lysates were obtained by homogenization of brains of from respective groups using 1x cell lysis buffer (provided in the kit). The protein concentration was determined for each sample and diluted to 300 μg in 1x blocking buffer. Samples for each group (three mice/group) were pooled and incubated with array membrane overnight at 4 °C. After washes, the membranes were incubated with biotin conjugated primary antibody provided in the kit overnight at 4 °C and next day following successive washes, membranes were incubated with secondary antibody provided for 2 h at room temperature. As per the ray biotech protocol, protein levels were visualized by chemiluminescence and quantified using ImageJ software. The protein levels on each array were standardized against an internal positive control on the array .As described previously , paraffit test and comparisons between multiple groups were done using one-way ANOVA followed by Tukey’s post-hoc multiple comparison test. Statistical significance was defined when ***p < 0.001, **p < 0.01, *p < 0.05. In all experimental procedures, n (per group) = 5–10 animal.The data quantified and represented as mean ± SEM. Statistical analysis was carried using Prism 7 . Comparison between two groups was achieved by unpaired The presence of pathological TDP-43-positive inclusions in cytoplasm and nucleus of both the neurons and glia in ALS, FTLD, and Alzheimer’s has been widely established , 24. MorThe main histological feature of neurodegenerative disorders such as ALS/FTLD and to a lesser extent AD is a presence of ubiquitinated TDP-43 immunoreactive cytoplasmic inclusions in neuronal and glial cells. In order to determine if cerebral ischemia increases the level of ubiquitination of TDP-43 in an age-dependent manner, we performed a double immunofluorescence analysis using anti-TDP-43 and anti-ubiquitin antibodies on 3- and 12-month-old control mice and 72 h after MCAO. As shown in Fig. Microglial activation coupled with a marked induction of Toll-like receptor 2 (TLR2) are characteristic features of the brain response to ischemic injury . Our preWe previously demonstrated that deregulation of TDP-43 observed in a mouse model of ALS/FTDL potentiates NF-κB-mediated pathogenic pathways . We showWe next investigated to what extent the observed age-related deregulation of cytokine response after stroke affects evolution of the post-stroke ischemic injury. As previously described  and showPrevious evidence suggests that cytoplasmic mislocalization of TDP-43 and formation of ubiquitinated aggregates is toxic to neurons and may enhance neuroinflammation , 24, 35.At present, it remains unclear to what extent and/or whether TDP-43 pathology is associated with ischemic injury in human stroke. To address this issue, we performed immunohistochemistry analyses of the post-mortem post-stroke brain tissue autopsied at 1 to 5 days after stroke. The analysis was performed by focusing on two distinct region of the ischemic lesion, the peri-infarct and core region  and compared with corresponding controls. The analysis was performed using a human anti-TDP-43 antibody Fig. a, b. As The work presented here provides an important in vivo evidence for a pathogenic role of TDP-43 in stroke. Based on the results presented in this study, we propose here that age-related deregulation of TDP-43 exacerbates inflammation and ischemic injury and may contribute to post-stroke neurodegenerative processes. By investigating TDP-43 expression patterns after stroke in young and 12-month-old mice, we showed (i) a marked increase and accumulation of TDP-43 in cytoplasmic compartment in neurons and microglia (ii) levels of mislocalized/cytoplasmic TDP-43 and its cleaved pathogenic fragments TDP-35 and TDP-25 were more elevated in aged mice, (iii) observed deregulation of TDP-43 was associated with an increase in post-stroke inflammation and larger infarctions, (iv) overexpression of TDP-43 further exacerbated ischemic injury and markedly enhances inflammation via activation of NF-κB, and (v) mislocalization of TDP-43 into cytoplasmic compartment occurred also in human stroke.Cerebral ischemia is characterized by a marked acute and chronic inflammatory response. We and others have shown that post-stroke inflammation may have a marked chronic component that may last several months following an initial ischemic event and may contribute to development of the chronic brain injury , 36. HowTo date, little is known about the role of TDP-43 in the pathogenesis of stroke. The alterations in TDP-43 expression in response to ischemic injury have been recently described by Kanazawa and colleagues in an acute rat model of ischemic injury . HoweverHere, we presume that cytoplasmic accumulation of TDP-43 during ischemic stroke occurred because of deregulation of its nuclear import. In fact, the C-terminal fragments of TDP-43 that are formed during aging/stress lack functional nuclear localization signal . Cytoplaproof-of-concept experiments using TDP-43 A315T transgenic mice that overexpress TDP-43 in the cytoplasm, at the similar levels as 12-month-old WT mice, we observed comparable levels of nuclear phosphorylation levels of P65 subunit of NF-κB after stroke.Another hallmark of the brain response to ischemic injury and neurodegeneration is activation of glial cells. Indeed, previous studies reported TDP-43 cytoplasmic inclusions glial cells in the spinal cords of the ALS patients  while inAn important question here is how deregulation of TDP-43 affects neuronal survival and microglia-neuron crosstalk after ischemic injury. Substantial loss of nuclear TDP-43 in ischemic neurons may lead to nuclear dysfunction. Indeed, a series of in vitro experiments showed that TDP-43 depletion/silencing may cause disturbance in cell cycle leading to cell death . AdditioIn conclusion, ischemic injury is associated with a marked and age-related deregulation of TDP-43. Further, we observed a significant increase in TDP-43-mediated modulation of NF-κB neuroinflammation leading to increase in neuronal injury and neurodegeneration after stroke. Although the stroked tissue did not display the full neuropathological feature associated with chronic TDP-43 proteinopathies, i.e., the presence of highly phosphorylated TDP-43 aggregates and complete depletion of nuclear TDP-43, we showed that ischemic injury may cause a long-lasting cytoplasmic accumulation of TDP-43. Thus, it is possible that deregulation of TDP-43 observed after the initial ischemic event may drive chronic post-stroke inflammatory response and may represent an age-related risk factor for development of neurodegenerative disorders. Based on our results, we propose that therapies targeting mislocalized/cytoplasmic TDP-43 may have a potential to attenuate post-stroke inflammation and ischemia-induced neuronal injury.Additional file 1:Table S1. Details and pathological features of the stroke subjects presented in Fig.


A 1 × 1 cm2 SiO2/Si substrate square was almost fully covered with graphene within 5 min, resulting in a record growth rate of ~33.6 µm/s. This outcome is attributed to the quick pyrolysis of methanol, with the help of trace copper atoms. The as-grown graphene exhibited a highly uniform thickness, with a sheet resistance of 0.9–1.2 kΩ/sq and a hole mobility of up to 115.4 cm2/V·s in air at room temperature. It would be quite suitable for transparent conductive electrodes in electrophoretic displays and may be interesting for related industrial applications.To realize the applications of graphene in electronics, a large-scale, high-quality, and uniform graphene film should first be placed on the dielectric substrates. Challenges still remain with respect to the current methods for the synthesis graphene directly on the dielectric substrates via chemical vapor deposition, such as a low growth rate and poor quality. Herein, we present an ultrafast method for direct growth of uniform graphene on a silicon dioxide (SiO T. T2 SiO22/Si substrates without further annealing, unless addressed in other parts in this paper. The species used for TEM tests were from the transferred graphene on the copper grids.The as-grown graphene was characterized using optical microscopy , transmission electron microscopy , Raman spectroscopy , and atomic force microscopy . The species that were used for the Raman, optical, and AFM tests were the as-grown graphene on the original SiOL) and width (W) were measured to be ~27 μm and 70 μm, respectively. To obtain better contact, the devices were thermally annealed at 200 °C in an H2/Ar (10/90 sccm) atmosphere for 30 min. The electrical measurements were carried out in air at room temperature using a semiconductor analyser .The electrical properties of the graphene were evaluated based on a field effect transistor (FET) configuration. The as-grown graphene film was first patterned into micro-ribbons. Then, Cr/Au metal contacts (10/50 nm) were fabricated onto the micro-ribbons via thermal evaporation to form the bottom-gate FETs. The channel length , carbon monoxide (CO), carbon dioxide (CO2), water and methane [2, is saturated at ~68.5% [2, will gradually reduce to CO and H2O due to the H2, leading to the final content of CO reaching up to ~25.5%. The residual CO2 and methane were below 5.5% and 0.5% in the composition, respectively, which was reasonably negligible in our experiment. Finally, the reaction of CO + H2→C + H2O occurred at our set temperature  with the copper catalyst, thus leading to highly active deposition of carbon radicals [2/Si substrate at the low energy locations and formed the carbon nuclei and domains as prolonging the duration of the process  revealed the degree of the defects and the in-plane crystallite size (La). The parameters that are derived from Raman spectra are accumulated in 2 gas. The value of ID/IG increased from 0.94 ± 0.30 to 1.79 ± 0.67 2 = (1.8 ± 0.5) × 10−9 × λL2(ID/IG)−1) [2 flow. In addition, the ratio of the 2D band over the G band (I2D/IG) gradually decreased from 1.74 ± 0.43 to 1.01 ± 0.77 −1)  reduced 1 ± 0.77 f. Previographene ,30. In o2/Si substrate by varying the growth duration. The optical images of graphene films that were obtained by various growth durations (0.5–30 min) are shown in 2 substrate, resulting in the limitation of further growth of the graphene flake. As a result, the numerous nuclei were responsible for our ultrafast growth rate. The substrate was almost fully covered by the graphene film within 5 min  in three-layer exfoliated graphene [The systematical Raman measurements revealed the quality, layer number, and defects of graphene samples with different growth durations . The val4 ± 0.11 a, reflecne oxide . Neverth samples ,34,35. T samples ,34,35. A6 ± 0.24 b. Simult0.24 /(ΔVgs·Vds·Cox), where, Cox is the silica gate capacitance (1.15 × 10−8 F/cm2 for a gate oxide thickness of 300 nm). As a result, the hole and electron mobility were approximately 115.4 and 13.7 cm2/V·s, respectively, which are higher than those of graphene film grown on some dielectrics  were made. The inset image in lectrics  [21,41,4lectrics . In addi2/Si substrate. The methanol precursor was rapidly catalytically decomposed once it was introduced into the tube, leading to the ultrafast nucleation and growth of the graphene and a record growth rate of ~33.6 µm/s. Meanwhile, the exclusion of H2 in the carrier gas reduced the etching of the as-grown graphene domains, thereby improving the crystallization of the graphene. As a result, the trilayer graphene film was of a good quality, with a sheet resistance of 0.9–1.2 kΩ/sq and hole mobility of up to 115.4 cm2/V·s in air at room temperature. These graphene films would be quite suitable for transparent conductive electrodes in the electrophoretic displays, which require moderate conductivity and mobility. Therefore, our method possesses a competitive advantage in related industrial applications. In summary, we have presented an ultrafast method for the direct growth of uniform graphene film on a SiO


The indicated structure and heating performance of the membrane, as well as the variation induced by Joule heating, would drive its applications.Nanofibrillated cellulose (NFC) and graphene oxide (GO) with reinforcing and film-forming properties were employed with graphene to develop a novel and thin electric heating membrane with heat dissipation controllability. A negative charge was found on the surface of GO and NFC in aqueous dispersions, which contributed to the homogeneous distribution of the graphene sheets. The membrane had a good laminated structure with three-dimensional interaction between GO and NFC, with embedded graphene sheets. Conductivity was characterized as a function of the amount of graphene, thus giving control over to the heating power by adjusting the ratio of graphene. Subsequent electric heating tests can remove irregularities on the GO and graphene sheet, improving the laminated structure further. The temperature on the surface of the membrane presented an exponential increasing regularity with time. Under the same power density and time, the stabilized temperature rise of membranes was higher when grammage was higher, which was characterized by the linear function of the power density. Low-grammage membranes (1 and 4 g·m It was also reported that there was a good linear relation between voltage and electric current, and a high coincidence degree was observed after 50 tests [Graphene-based materials such as graphene nanosheets, multilayer graphene nanoplatelets, graphene oxide (GO), and reduced graphene oxide (RGO) are currently of considerable interest in many fields, including composite materials, paints and coatings, flexible electronics, energy generation and storage, sensors and metrology, and bioapplications ,2,3. In 50 tests . In addi−1 [However, this Joule heating effect has more diverse applications than simply heating. When RGO is used for current collection in a lithium battery, after Joule heating up to 2750 K for 1 min by applying a direct current, the sheet resistivity has been demonstrated to drop as low as 0.8 Ω/sq, with conductivity of up to 3112 S·cm−1 .It is clear that endowing graphene and graphene-based composites with electric heating functions has much promise; however, there are few systematic studies focusing on the electric heating performance. While polymer substrates such as polydimethylsiloxane  and epoxConsequently, a new strategy was developed for fabricating NFC-graphene-GO 3D systems for electric heating membranes. A mixture of NFC and GO was first prepared, which acts as a binder and dispersion stabilizer, before the graphene sheet dispersion was added. In addition, the influence of the ratios among graphene, NFC, and GO on the morphology, structure, conduction, and heating performance are systematically reported in this work, as well as the effect induced from Joule heating. Moreover, the relationship of grammage to conductivity and heating performance will be taken into account, aimed to produce a thinner membrane with suitable power efficiency. This presented work endowing the membrane with a new-type of electric heating function would further broaden the application of NFC–graphene based composite.−1, pH 5–7, specific surface area 1000–1217 m2·g−1, thickness 0.6–1.0 nm, and a lamellae diameter 0.5–5 μm, was from Suzhou TANFENG Graphene Tech. Co., Ltd., Suzhou, China; graphene, was produced by the CVD method, 1–2 layers, purity > 99 wt.%, and a specific surface area of 1000–1217 m2·g−1, sheet thickness 0.5–3.0 nm, lamellae diameter 0.5–5 μm, was produced by Suzhou TANFENG Graphene Tech. Co., Ltd., Suzhou, China; copper foil electrodes with the a thickness of 0.03 mm and width 5 mm.NFC dispersion, concentration 1.07 wt.%, diameter 10–20 nm, length 2–5 μm, was manufactured by Tianjin Woodelfbio Cellulose Co., Ltd., Tianjin, China; GO dispersion, concentration 2 mg·mLThe process for producing NFC–graphene–GO composite membranes is summarized in Next, a graphene dispersion was prepared by mixing a certain amount of graphene with 20 mL of distilled water. The mixture was stirred using a magnetic stirrer for 5 min, and then treated with ultrasonication .Finally, a NFC-graphene-GO dispersion was formed by pouring the NFC-GO mixture into the graphene dispersion. The mixture was treated with ultrasonication  and filtered with a 0.22 μm organic filter . The dispersion was then placed in a vacuum drying oven at 60 °C for 12 h to obtain NFC-graphene-GO composite membranes, which were kept in an indoor environment (about temperature of 20 °C and humidity of 60%) for 24 h.−2 (dry weight); the grammage was varied at 1, 4, 8, 12 and 16 g·m−2 with a constant 50 wt.% graphene. Further composite membranes were prepared to investigate the influence of GO on the surface morphology and phase structure. The GO contents were 5, 15, 25, and 30 wt.%, with a constant 50 wt.% graphene, corresponding to GO to NFC weight ratios of GO:NFC = 1:9, 3:7, 1:1, and 3:2.Composite membranes were prepared with different grammages or graphene contents, in which the weight ratio of GO to NFC was kept constant, at 1:1. The graphene content was varied from 20 to 55 wt.% in 5 wt.% intervals with a controlled grammage of 16 g·m2, to which copper foil electrodes were fixed at both ends, to prepare electric heating membranes with an effective heating surface area of 20 × 30 mm2. Electric heating device and installation of electrodes and temperature senor on the membrane can be seen in Composite membranes with different amounts of graphene and grammages were used to prepare electric heating membranes. First, a four-probe resistivity tester  was used to test the sheet resistance of the membranes at 7 equidistant points , with thChange rate of resistance between two electrodes of the membrane after different bending cycles (bending angle of about 90°) of 0–600 times at 100 times intervals were tested for the evaluation on its stability and flexibility using a multimeter . The change rate is the percentage of the absolute value of difference between the resistance before and after bending to the resistance before bending.−2 was applied to each membrane for 15 min with a voltage regulator controlling the power, considering the tested resistance between the two electrodes and the effective surface . In both tests, the temperature was recorded every 2 s at two test points using a multichannel temperature recorder ; the room temperature was simultaneously recorded to calculate the temperature rise . The resistance between the two electrodes was recorded before the heating test, at the outage moment, and when cooling to room temperature, to investigate the membrane’s power stability.To improve the stability of the heating performance, membrane annealing was induced by Joule heating ,12 in a −1, ultrasonic treatments at 1000 W for 5 min, then 800 W for 20 min), and NFC–graphene–GO dispersion , ultrasonic treatment as for the composite membrane above) were analyzed in an aqueous solution using transmission electron microscopy  at 100 kV. Both the surface and cross-section were observed using scanning electron microscopy . The zeta potential and particle size of NFC, GO, and NFC-GO dispersions were analyzed in aqueous solution with the Zetasizer Nano ZSP . The concentrations of the NFC and GO solutions were 0.25 mg·mL−1, and that of the mixed dispersion was 0.5 mg·mL−1. The 3D surface morphology and roughness (10 × 10 μm area) were analyzed using atomic force microscopy  in tapping mode. Fourier transform infrared (FT-IR) spectra of powdered samples were collected from 400 to 4000 cm−1 with a Nicolet iS50 spectrometer  that had a resolution better than 0.09 cm−1. The phase structure of the membrane was analyzed by laser confocal micro-Raman spectrometry  from 4000 to 500 cm−1 with an excitation wavelength of 532 nm. Each sample was tested three times. X-ray diffraction  was carried out for 2θ from 5° to 70° with an 8°·min−1 scanning speed on graphene, NFC, GO, and the composite membrane. Differential thermal-thermogravimetric analysis , Shimadzu Corp., Kyoto, Japan) was applied to study the thermal performance during heating from room temperature to 600 °C at a rate of 10 °C·min−1 in a nitrogen atmosphere, with respect to NFC, GO, graphene, and the composite membrane ). The surface morphology and phase structure of the membrane ) were evaluated after the two electric heating tests using SEM, Raman, and XRD, and compared with that before heating.The morphology of graphene, NFC, and GO dispersions  in the spectra were almost the same for graphene and the membrane (2.13 vs. 2.26), whereas both values were greater than that for GO (1.59) [D/IG values with a RGO membrane, as pure GO sheets with abundant active groups stack more regularly and combine more compactly when cast and annealed by Joule heating. The position of the G peak of graphene and the membrane were located around 1587 and 1586 cm−1, respectively, while the D peak shifted from 1357 to 1361 cm−1. The full width at half maximum (FWHM) of the D peak increased from 207 to 221 cm−1, and that of the G peak increased slightly from 81 to 87 cm−1, meaning that integrity of the lamellar structure of graphene sheets embedded with NFC and GO had been slightly deteriorated. Slight variation of the phase structure of membranes with different amounts of graphene and GO were also found, as shown in As shown in O (1.59) , indicatO (1.59) , who demθ = 25.8°, indicating the well exfoliated state of the graphene layers compared with that in graphite [θ = 40.5° was strengthened as the amount of graphene was increased, and was prominent in the membrane with 50 wt.% graphene  of the GO membrane (d as 0.819 nm according to Bragg’s diffraction formula [d spacing = 0.832 nm) and became gentle, and was indistinguishable as the amount of GO decreased to 5 wt.%, due to the disruption caused by the added NFC and graphene. Hence the ordered layers of GO, which include oxygen-containing groups, are expected to be partly diverged [The characteristic reflections . Thus, a temperature rise of 90 °C can be achieved by applying 2500 W·m−2 power density  in the spectrum, which decreased from 2.21 to 1.84. Likewise, the I2D/IG ratio declined slightly from 0.67 to 0.59, and the peak position shifted to a higher wavenumber. The FWHM of the D, G, and 2D peaks all decreased, from 216 to 188 cm−1, 86 to 81 cm−1, and 398 to 336 cm−1, respectively. The change in ID/IG values differed from those in published works; however, while previous studies focused on neat RGO reduced by Joule heat with high power [θ = 10.7° in the XRD pattern  results indicated that thermal stability of the membrane was improved due to the crosslinking of NFC and GO. The regularity of the membrane structure was elevated after two heating tests, with regard to the CRR at the moment power was lost and after cooling. The CRR showed suitable relationships with the grammage and graphene content, which will contribute to the control over the actual power in use. However, before further application, additional mechanisms and technologies must be investigated, such as the variation of phase structure induced by Joule heating with a wider range of power and longer working time, the relation between phase structure and thermal radiation, and key technologies for large scale printing or coating.


Over the last decade, the immune checkpoint blockade targeting the programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis has improved progression-free and overall survival of advanced non-small cell lung cancer (NSCLC) patients. PD-L1 tumor expression, along with tumor mutational burden, is currently being explored as a predictive biomarker for responses to immune checkpoint inhibitors (ICIs). However, lung cancer patients may have insufficient tumor tissue samples and the high bleeding risk often prevents additional biopsies and, as a consequence, immunohistological evaluation of PD-L1 expression. In addition, PD-L1 shows a dynamic expression profile and can be influenced by intratumoral heterogeneity as well as the immune cell infiltrate in the tumor and its microenvironment, influencing the response rate to PD-1/PD-L1 axis ICIs. Therefore, to identify subgroups of patients with advanced NSCLC that will most likely benefit from ICI therapies, molecular characterization of PD-L1 expression in circulating tumor cells (CTCs) might be supportive. In this review, we highlight the use of CTCs as a complementary diagnostic tool for PD-L1 expression analysis in advanced NSCLC patients. In addition, we examine technical issues of PD-L1 measurement in tissue as well as in CTCs. EGFR mutations or ALK rearrangements, and, in addition, factors predictive of response to immunotherapy, thus, immune checkpoint inhibitors (ICIs). The introduction of ICIs in the clinic has led to increasing response rates in locally advanced and metastasized NSCLC [Lung cancer is the most common cancer in men and the third frequent cancer in women worldwide. With a poor five-year survival rate of 10–15%, lung cancer is the major cause of cancer-related deaths [ed NSCLC ,3,4,5. Ied NSCLC . Therebyed NSCLC .ClinicalTrial.gov [ClinicalTrial.gov, 3% of studies with Nivolumab, 6% of studies with Atezolizumab, and 3% with Pembrolizumab treatment have been completed , underl+) CTCs in NSCLC is to date in its infancy as the first related study was published in 2016 by Schehr and colleagues [+ CTC enrichment. Schehr et al. reported the presence of a population of co-isolated CD11b+ (marker for myeloid development), CD45-low, and cytokeratin-positive (CK+) cells—via an in-house produced immunomagnetic enrichment system—misidentified as CTCs and expressing PD-L1. The quantification of PD-L1 could therefore be skewed by false positive events, thus requiring careful analysis in order to increase the accuracy of the assay [+ patients (~7% based on CK19 mRNA expression) did not allow for drawing any significant conclusion about the clinical utility of PD-L1 [Despite the reported value of assessing the overexpression of PD-L1 on cells of different types in solid tumors—including lung cancer—as a promising marker to predict anti PD-1/PD-L1 treatment efficacy, the predictive value of PD-L1 expression is still controversial and related investigations face the three major limitations of tissue biopsies: Invasiveness, sampling error due to tumor heterogeneity, and mostly unfeasible longitudinal sampling. To overcome these issues and support histological analysis, the expression of PD-L1 has been explored on CTCs and has been correlated to patients’ clinical outcomes . The clilleagues , who inihe assay . Howeverhe assay  also focof PD-L1 .+ CTCs in a chemotherapy setting has been further investigated by Kallergi and colleagues [+ and PD-L1+ CTCs in metastatic NSCLC patients before  and after chemotherapy . Giemsa staining revealed CTCs in 28 of 30 (93.3%) patients at baseline and in 9 of 11 (81.8%) patients studied after the third chemotherapy cycle with a median of 5 CTCs/mL of blood . Of interest, using immunostaining, CTCs could be detected in 17 of 30 (56.7%) patients at baseline and in 8 of 11 (72.7%) after the third treatment cycle. The concordance between the two detection methods at baseline and after the third treatment cycle was 63.3% and 67%, respectively. The rate of detection was 30% (9 of 30) and 27% (8 of 30) before treatment, and in 9% (1 of 11) and 46% (5 of 11) after 3 cycles of chemotherapy, respectively for PD-1+ and PD-L1+ CTCs. Interestingly, an increase of 20% PD-L1+ CTCs (p = 0.096) and a decrease of 21% PD-1+ CTCs (p = 0.785) after chemotherapy was observed. In addition, a shorter progression-free survival (PFS) could be observed for patients with >3 PD-1+ CTCs at baseline (p = 0.022) but not for PD-L1 expressing tumor cells, thus suggesting a potential clinical role for PD-1+ CTCs rather than for PD-L1+ CTCs [The predictive utility of PD-L1lleagues . In thisL1+ CTCs .Through a different size-based CTC-enrichment approach  followed by immunostaining, Adams et al.  investign = 5) or chemoradiation (n = 8). Serial blood samples from the patients enrolled in the study were collected before the initiation of radiation, during radiation, and at follow up, approximately one month after radiation (n = 38 samples). CTCs were detected in all 38 samples with an average of 21.3 CTCs/mL (range of 4–72 CTCs/mL), while PD-L1+ CTCs were detected in 24 (66.7%) out of 36 samples analyzed, ranging from 0 to 43 PD-L1+ CTCs/mL. In line with the results by Adams et al. [+ CTCs during treatment ). In addition, PD-L1−positive patients had a shorter PFS compared to PD-L1-negative patients using a PD-L1+ CTC cut-off ≥5%. Notably, one of the patients who had a high PD-L1+ CTC count at visit two and visit three was put on therapy with Pembrolizumab after initial progression and has had stable disease for seven months [The impact of radiation therapy on PD-L1 expression in CTCs was recently investigated by Wang et al.  monitoris et al. , patientn months , implyin+ CTCs in the clinical setting, were conducted by Ilié et al. [® System  followed by immunostaining of retained cells. In the study of Ilié and colleagues, CTCs were detected in 80 of 106 (75%) patient samples, while 99% (79 out of 80) CTC-positive samples exhibited more than 5 CTCs per 4 mL blood, with a median of 60 CTCs per 4 mL (range: 2–256 CTCs/4 mL). Furthermore, the researchers extracted ≥1 PD-L1+ CTCs in 8% of patients with advanced stage III and IV NSCLC (n = 6/71 samples) with 93% concordance to PD-L1+ tumor cells of matched primary tissue . In addition, they could observe a trend towards poor clinical outcomes in patients with PD-L1+ CTCs receiving first line of chemotherapy, similar to the trend observed for PD-L1+ primary tumors [n = 61/89 samples) and ≥3 CTCs in 33.7% (n = 30/89 samples) of NSCLC (mostly stage IV) patients using the ParsortixTM system. Thereof, the researchers found ≥1 PD-L1+ CTC in 56% (n = 50/89 samples) and ≥3 PD-L1+ CTCs in 26% (n = 23/89 samples) of patients. Amongst patient samples with at least three CTCs (CD45−/K+), 47% (14/30) harbored exclusively PD-L1+ CTCs and 47% (14/23) had both PD-L1+ and PD-L1− CTCs [+ CTCs did not correlate with the percentage of PD-L1+ tumor cells in primary tumor tissue biopsies determined by immunohistochemistry (p = 0.179). In patients undergoing therapy with Pembrolizumab, Nivolumab, or Atezolizumab the researchers indicated that in 89% of the responding patients either a decrease or no change of their total CTC counts after three or five cycles of therapy  was shown. In contrast, upon disease progression, all patients showed an increase in PD-L1+ CTCs [The largest studies so far, investigating the role of PD-L1é et al.  and Janné et al. , who utiy tumors . JanningL1− CTCs . Of inteL1+ CTCs . + CTCs in NSCLC patients under immunotherapy has also been investigated in several other studies. Nicolazzo and colleagues [+ CTCs utility in patients with stage IV NSCLC treated with the anti-PD-1 Nivolumab. By utilizing the CellSearch® system as a CTC enrichment approach, they reported a CTC detection rate of more than the 40% usually described by the literature [+ CTCs ranged from 25% to 100%, while after six months 50% showed PD-L1+ CTCs. Therefore, even though both the presence of CTCs and the PD-L1 expression were associated with poor clinical outcomes (statistics not available), the lack of patients with PD-L1-negative CTC fractions did not allow for any conclusions about the real prognostic and predictive relevance of this marker [+ CTCs in 93% of advanced NSCLC patients before Nivolumab treatment, with a median proportion of CTCs expressing PD-L1 of 17.2% using the ISET technology followed by immunostaining. Interestingly, no correlation could be observed with PD-L1+ tissue biopsies . Furthermore, in a study by Kulasinghe and colleagues [+ CTCs —enriched through the size-based ClearCell FX—were treated with Nivolumab, but no correlation between PD-L1 expression and clinical outcomes could be observed. The impact of the epithelial-to-mesenchymal transition (EMT) of CTCs in NSCLC patients under Nivolumab treatment was described in a recent study by Raimondi et al. [+ CTCs. Interestingly, PD-L1 was found coexpressed with EMT markers in a percentage of cells that was ranging between 50% and 78%. This might provide a biologic explanation for the persistence of PD-L1-positive CTCs in NSCLC patients after six months of treatment, predicting resistance to the anti-PD-1 Nivolumab shown by Nicolazzo and colleagues [The predictive value of PD-L1lleagues  first foterature , with ans marker . In addis marker  detectedlleagues , 66% of i et al.  using thlleagues .n = 4), statistical analysis was not possible. Importantly, patients with >50% PD-L1+ CTCs (n = 3/4) experienced an improved progression-free survival under Pembrolizumab treatment, indicating the need for further confirmation of the available data to reconcile the conflicting evidence.Beside the potential predictive role of PD-L1+ CTCs for Nivolumab treatment, its clinical significance has also been investigated for immunotherapy based on Pembrolizumab (PD-1 inhibitor) by Dhar and colleagues , who alsIn summary, despite the limited amount of studies published to date, these first results imply PD-L1+ CTCs might play a role in determining response to different ICI therapeutic approaches (summarized in + or PD-1+ tumor-infiltrating lymphocytes (TILs) were significantly associated with prolonged disease-free survival of these patients, whereas PD-L1 and PD-L2 expression had no significant prognostic implications. He and colleagues [Several studies showed that the efficacy of PD-1/PD-L1 blockade could be also affected by PD-L1 expression on tumor-infiltrating cells in different types of cancer, including lung cancer. Herbst et al.  showed alleagues  revealedHowever, it has to be noted that even though the field of CTCs carries a great potential with liquid biopsy to better integrate the heterogeneous and potentially dynamic expression of PD-L1 in the course of NSCLC pathology, it still requires a series of (pre-)analytical standardizations, in order to avoid misinterpretations and guarantee a reliable clinical treatment decision . Important aspects need to be taken into consideration when comparing these studies. First and foremost, a detailed report about patients´ clinical data  followed by more technical standardization regarding the sampling, blood stabilization, storage time and temperature, and CTC enrichment and detection approach, as well as the antibody cocktail utilized for the immunostaining and the threshold applied for the PD-L1 positivity are necessary . Indeed,In order to address this need for liquid biopsy—including CTC—standardization, several public–private partnerships and consortia were established: BloodPAC, European Liquid Biopsies Academy, Liquid Biopsy Consortium, SPIDIA4P, and the European IMI CANCER-ID consortium currently addressing—among others—the PD-L1 harmonization issue to detect PD-L1+ CTCs in NSCLC. In a collaboration project within the CANCER-ID consortium, we performed a comprehensive multicomparison of commercially available anti-PD-L1 antibodies in a NSCLC cell line panel including a preincubation with Atezolizumab (manuscript in preparation). Indeed, one of the major issues in translating PD-L1+ (circulating) tumor cells from basic research to the clinical setting for routine diagnostic application is (i) a heterogeneous detection rate of used CTC enrichment and detection approaches, (ii) resulting observer bias in calling CTCs, and (iii) the lack of consensus on the use of different commercially available anti-PD-L1 antibody clones and their performance and specificity compared to the antibody clones that are included in the IHC kits that received regulatory approval. ® System which is FDA approved for clinical utility in metastasized breast, prostate, and colorectal cancer, remains challenging in NSCLC and studies using this system have to view with caution. To overcome the low sensitivity in advanced NSCLC patients using the CellSearch® System, alternative methods for CTC detection were used in most studies  for the assessment of PD-L1 expression of CTCs extracted from NSCLC patients. They showed a 50% higher detection rate of CTCs per blood sample with the ParsortixTM system (>1 CTC in 59 of 97 samples (61%) compared to 31 of 97 samples (32%) with >1 CTC using the EpCAM-based system). Another promising method is the filter- and size-based ISET system which was used in several recent CTC-related studies in NSCLC patients  than by the CellSearch® system . However, statistical significance was not reached in SCLC, though the trend favoring the MCA system over the CellSearch® system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch® system [Using the ISET method, Farace et al.  showed c® system . Since most PD-L1 CTC studies use different enrichment techniques and methods for CTC detection, it becomes clear that comparisons between these studies  and tremelimumab (anti-CTLA-4) (NCT03275597) uses CTC number and CTC PD-L1 expression as exploratory endpoints for efficacy and target engagement. Future research will show whether CTC PD-L1 expression together with additional biomarkers like tumor mutational burden assessed by the analysis of CTCs or ctDNA, constitute clinically relevant blood-based biomarkers for immune checkpoint blockade therapy patient selection.Immune checkpoint inhibition therapy represents a breakthrough in treatment of non-small cell lung cancer patients. However, there are still major challenges in selecting NSCLC patients likely to benefit from targeting the PD-1/PD-L1 axis. CTC-based liquid biopsy may be an option for the development of blood-based tests that address this issue. The successful implementation of such tests will critically depend on consensus on the use of different anti-PD-L1 antibody clones, CTC enrichment technologies, and well-established standardized clinically feasible standard operating procedures. Furthermore, a deeper understanding of the different mechanisms of PD-L1 regulation at genetic, epigenetic, transcriptional, translational, and posttranslational levels in cancer is needed to develop appropriate protocols. In addition, the regulation of PD-L1 expression during metastasis might be different from the primary tumors, potentially making longitudinal monitoring of patients necessary and liquid biopsy an even more favorable diagnostic option. The latter is further supported when taking into account that PD-L1 expression assessed by IHC requires a tissue sample which could be insufficient, or even lacking, in advanced lung cancer patients. This may compromise the level of confidence with which a therapy decision can be made. Several studies suggest a promising role for PD-L1


For all eight lesion types, the highest burden was observed for pTDP-43. In six of the eight lesions studied, cTDP-43 burden was greater than nTDP-43 burden. However, we observed a higher burden of nTDP-43 to cTDP-43 for pre-inclusions. We also noted an equal-to-slightly higher burden of nTDP-43 to cTDP-43 for peri-vascular inclusions. There was not strong evidence for associations to be driven by mutation status although for neuronal cytoplasmic inclusions and dystrophic neurites GRN+ cases had higher burden of pTDP-43, cTDP-43 and nTDP-43 compared to GRN- cases, with nTDP-43 inclusions only observed in GRN+ cases. In addition, for pre-inclusions, cTDP-43 and nTDP-43 burden tended to be higher in C9ORF72- cases compared to C9ORF72+ cases, although this was not the case for pTDP-43. There is clear evidence that phosphorylation and C terminal fragments play an important role in lesion formation in FTLD-TDP. However, for some inclusions, particularly pre-inclusions, full-length TDP-43 appears to be playing a role.The transactive response DNA binding protein of 43 kDa (TDP-43) is an intranuclear protein involved in RNA splicing. Abnormally deposited TDP-43 is found in the brains of patients with frontotemporal lobar degeneration (FTLD). Different morphological characteristics of TDP-43 immunoreactive inclusions define the different variants of FTLD-TDP. Little is known about the relationships between TDP-43 specie  and lesion types. Using novel antibodies that recognize phosphorylated TDP-43 (pTDP-43), a neoepitope in the C-terminal fragment of TDP-43 (cTDP-43) and the N-terminal, i.e. full length (nTDP-43) we assess the relative burden of pTDP-43, cTDP-43 and nTDP-43 in 8 different lesion types across FTLD-TDP type A-C. These include neuronal cytoplasmic inclusions, dystrophic neurites, neuronal intranuclear inclusions, fine neurites of the hippocampus, peri-vascular inclusions, Pick body-like inclusions, long thick dystrophic neurites and granular pre-inclusions. We also assess for associations with  GRN) gene . To assess for the presence of an expanded GGGGCC hexanucleotide repeat in C9ORF72, the repeat primed PCR was also used, as outlined in a previous publication [In all 24 cases we screened for the presence of a mutation in the lication .GRN gene and one had a mutation in the C9ORF72 gene. Of the eight FTLD-TDP type B cases, four had a mutation in the C9ORF72 gene. The Thal β-amyloid phase and the Braak NFT stage were both low and relatively similar across FTLD-TDP types; none were Braak NFT stage IV or greater and none met intermediate - high probability criteria for Alzheimer’s disease [Demographic characteristics of the 24 cases are shown in Table  disease . HippocaThere were no observable differences in lesion burden between the entorhinal cortex, CA1 sector and subiculum of the hippocampus, and hence these regions were combined for further analyses. In all instances the pTDP-43 antibody detected the greatest number of inclusions compared to cTDP-43 and nTDP-43 (Table In order to better understand the findings regarding the burden of pTDP-43, cTDP-43 and nTDP-43 that we observed, and to allow us to see whether the findings may be driven by any one or more lesion type, we compared pTDP-43, cTDP-43 and nTDP-43 burden for each of the six lesion types. In the entorhinal/CA1/subiculum Fig. , we obseIn the dentate nucleus of the hippocampus Fig. , the pTDC9ORF72+ cases were the youngest at death. The clinical diagnoses, and β-amyloid and NFT distribution, were relatively similar between GRN+ and GRN- cases and between C9ORF72+ and C9ORF72- cases. Hippocampal sclerosis was present in all GRN+ and GRN- cases and 50% of C9ORF72 + cases, but was not present in any of the C9ORF72- cases. In all instances the pTDP-43 antibody detected the greatest number of inclusions compared to the cTDP-43 and nTDP-43 antibodies. There was little evidence for striking differences in the burden of lesions detected with the pTDP-43, cTDP-43 and nTDP-43 antibodies by mutation status. The higher proportion of lesions detected with nTDP-43 vs cTDP-43 antibody in the entorhinal/CA1/subiculum in FTLD-TDP type B cases was similar for C9ORF72+ and C9ORF72- cases.Demographic characteristics of the 16 FTLD-TDP type A and B cases by mutation status are shown in Table GRN status within FTLD-TDP type A cases, and C9ORF72 status within FTLD-TDP type B cases, for each lesion type (Fig. In order to get a better understanding of whether mutation status may be playing a role in the relationships we observed between the different antibodies and lesion type, we compared pTDP-43, cTDP-43 and nTDP-43 antibody burden for each lesion type, by mutation status. Hence, we compared GRN+ cases compared to GRN- cases for small discrete NCI’s, DNs and fine neurites. We also observed that lesion burden was higher for the nTDP-43 antibody in GRN+ cases compared to GRN- cases for small discrete NCIs, DNs and perivascular inclusions. In fact, the GRN- cases had no nTDP-43 small discrete NCIs or DNs. Comparing across antibodies, there was a higher burden of lesions detected with the cTDP-43 antibody than the nTDp-43antibody in most instances, except for with perivascular inclusions.First we describe our findings for FTLD-TDP type A associated lesions. We found that lesion burden was higher with the pTDP-43 antibody for GRN+ cases compared to GRN- cases for all lesions except for fine neurites where burden was about equal. We noted that lesion burden was higher for the cTDP-43 antibody in C9ORF72+ cases had a lower burden of lesions for both nTDP-43 and cTDP-43 antibodies compared to C9ORF72- cases. In fact, we did not see any C9ORF72+ cases with pre-inclusions that were detected with the cTDP-43 antibody. We also found that the C9ORF72+ cases had a slightly higher burden of pre-inclusions that were detected with the pTDP-43 antibody compared to the C9ORF72- cases. Comparing across antibodies, we observed that our finding of higher lesion burden for the nTDP-43 antibody compared to cTDP-43 antibody was observed for both C9ORF72+ and C9ORF72- cases.For FTLD-TDP type B we investigated mutation status for pre-inclusions. We found that the The results of this study demonstrate that phosphorylated TDP-43, C terminal specie and full length TDP-43 are all present in the inclusions in FTLD-TDP. Given that the characteristics of the different FTLD-TDP variants are dependent on different inclusion types, the results of this study support the notion that there are indeed differences in TDP-43 specie across the different FTLD-TDP variants. The findings improve our understanding of the relationship between TDP-43 specie and FTLD-TDP and may shed light on the cellular mechanisms resulting in lesion formation in the different FTLD-TDP types, with the findings suggesting different underlying mechanisms in FTLD-TDP type B. This knowledge may be helpful for the development of future treatments targeting TDP-43.Previous studies have demonstrated that the inclusions found in FTLD-TDP consist of phosphorylated TDP-43 , 17, 33.C9ORF72 gene and the clinical diagnosis was FTD-MND. This case, however, was classified as FTLD-TDP type A given the presence of fine neuritis in the CA1 region of the hippocampus [The burden of inclusions detected with the cTDP-43 antibody was higher than the burden of inclusions detected with the nTDP-43 antibody for most inclusion types, including fine neurites of the hippocampus, perivascular inclusions, small discrete NCIs and both short/comma and long thick DNs. These inclusion types, therefore, appear to consist mainly of C terminal TDP-43 and concur with previous studies that have also noted a predominance of C terminal TDP-43 in FTLD-TDP inclusions , 21. A npocampus , 25. It We also noted that perivascular inclusions in FTLD-TDP type A cases were slightly more likely to be detected with the nTDP-43 antibody compared to the cTDP-43 antibody. This trend was not as striking as for pre-inclusions, but was very different from the findings across the other inclusions identified in FTLD-TDP type A. In fact there was a case where no perivascular inclusions were detected with the cTDP-43 antibody while they were detected with the nTDP-43 antibody. In this case the pTDP-43 antibody also detected the perivascular inclusions. This finding would support the notion that cellular mechanisms resulting in lesion formation may differ within FTLD-TDP type A. Once again previous studies that have assessed C terminal fragments and full-length TDP-43 across FTLD-TDP types have not assessed perivascular inclusions , 15. SimWhen we studied NIIs we observed that the highest lesion burden occurred with the pTDP-43 antibody of which only a subset was identified with the C terminal antibody; none were detected with the nTDP-43 antibody. This finding is different from that reported in one previous study where the investigators found evidence for both C terminal specie and full length TDP-43 to be a component of NIIs . Our finWe should also point out that when we identified inclusions with the nTDP-43 antibody we also noted there was an absence of normal full-length TDP-43 from the nucleus of the associated cells. This implies that full length TDP-43 that is sequestered into the inclusion was unable to shuttle back into the nucleus. Whether this is due to the phosphorylation of the full length TDP-43, given that the pTDP-43 antibody detected the inclusions, or whether it is due to another type of post translational modification, remains unknown.GRN mutations to be associated with TDP-43 fragmentation [GRN+ cases showed a higher burden of TDP-43 inclusions, independent of the specific antibody, with only a few exceptions. The only demographic difference between the GRN+ and GRN- cases was a younger age in the GRN+ cases. Previous studies have shown that GRN mutations are associated with a more aggressive disease [C9ORF72 mutation. Interestingly, we noted that the C9ORF72+ cases had a slightly higher burden of pre-inclusions that were detected with the pTDP-43 antibody compared to the C9ORF72- cases while the C9ORF72- cases had a higher burden of inclusions with the cTDP-43 and nTDP-43 antibodies, suggesting that there were phosphorylated pre-inclusions in the C9ORF72+ cases that must not have been detected with either the cTDP-43 or nTDP-43 antibodies. This is not necessarily surprising given that other C terminal fragments have been reported in FTLD-TDP [Our analysis by FTLD mutations showed that our general findings were not driven by mutation status in any of the FTLD-TDP types. This is not surprising as animal models have not found any evidence for entation , 31 and  disease , and our disease , 29. We FTLD-TDP , 40, 44.GRN or C9ORF72 mutation. Despite the fact that the number of cases assessed was not large enough to allow for statistical testing, we have identified some interesting trends in the data, particularly in the pre-inclusions in FTLD-TDP type B, which warrant further investigation. Furthermore, the findings have relevance in the interpretation of studies from different laboratories as it is clear that some inclusions may not be seen depending on what kind of anti-TDP-43 antibody is being used for detection.A strength of this study was the fact that we looked at eight different inclusions and the fact that all patients underwent genetic screening for a


In this paper, a new electro-optical switch modulator based on the surface plasmon polaritons of graphene is proposed. An air–graphene-substrate–dielectric structure is adopted in the modulator. In this structure, the graphene is considered as a film of metal whose thickness tends to be infinitesimal. By changing the external voltage, the boundary conditions can be changed to decide whether the surface plasmon polariton waves can be excited in mid-infrared band. Because of this effect, the structure can be used as an electro–optical switch modulator, whose modulation depth is about 100% in theory. Finally, the 3 dB bandwidth (~34 GHz) and the energy loss (36.47 fJ/bit) of the electro–optical switch modulator are given, whose low energy loss is very suitable for engineering applications. Surface plasmon polaritons (SPPs) ,2,3,4,5 2 bonds. For this crystal structure, graphene has excellent electrical and optical properties. Specifically, in a wide spectral range, the optical absorption rate of graphene with intrinsic monolayer thickness is 2.3%, which is 50 times the same thickness as gallium arsenide. Based on the strong absorption effect of graphene on light, the light absorption can be adjusted by changing the voltage [Graphene ,14,15,16 voltage  and creaAn electro–optical switch ,19,20,21The mid-infrared band is a vital band, which has important application in different fields. For example, it can be used in sensing, environmental monitoring, and other places. Optoelectronic devices have many advantages, which include the greater plasma dispersion effects and the ease of manufacture in current technology.Many researchers have used the tunability of graphene to achieve electro–optical modulators, but the large volume and low modulation depth become the common problem for these modulators. To solve these problems, an air–graphene-substrate–dielectric structure is proposed. It makes use of the tunability of graphene and realizes the transmission or cutoff of the medium exciter wave in the whole structure. Theoretically, this modulator can achieve the modulation depth of 100%, which cannot be obtained by the traditional electro–optical modulator. Finally, the 3 dB bandwidth of the electro–optical switch modulator and energy loss of the switch state are also given. The modulator proposed here is very suitable for engineering application because of the low energy loss.The new electro–optical switch modulator is composed of an air–graphene-substrate–dielectric structure. Concretely speaking, it uses graphene-substrate as the thin film metals between the interface of air and dielectric . And for the electrode Au, it is placed in the position shown in This design can change the electrical conductivity of graphene by changing the voltage, to regulate the transmission and cutoff of surface plasmon polaritons waves. Finally, the aim of electro–optical modulation is achieved.The principle involved in this paper is mainly divided into two parts. One part is the change of the relationship between voltage and the graphene photoconductivity. Another part is the excitation of the graphene material to the dielectric interface surface plasmon polaritons waves.The relationship between voltage and graphene photoconductivity can be obtained by using the relationship between the chemical potential (Fermi level) and the cooper equation obtained from the plate model.0 can be given by Equation (1).Q is the charge of the capacitor, e indicates the amount of electron charge and the size is 1.6 × 10−19 C. S represents the area of the plate capacitor. And the Q can be given by Equation (2).V refers to the voltage applied externally. V0 is related to the Fermi level of graphene when it is not applied to voltage, which is usually to take 0 V.For the graphene, it’s carrier concentration nħ represents the reduced Planck’s constant whose size is about 1.055 × 10−34 J·s. The fV indicates the Fermi velocity of graphene, which is 1.1 × 106 m/s. ε0 refers to the dielectric constant in free space, and rε is the relative dielectric constant of the substrate material. d represents the thickness of graphene and substrate.Among them, w refers to the angle frequency of incident light. τ1 and τ2 indicate the intra-band and inter-band lag time of graphene. Here it is taken 10 fs and 1.2 ps respectively. In addition, the σ0 is equal to πe2/2h.Among them, By this way, the relationship voltage and the conductivity of graphene can be obtained.The boundary conditions of Maxwell equations are given in Equation (5).ρ represents the volume charge of the medium interface, and sJ indicates the surface current size of the medium interface.In Equation (5), The graphene can be seen as a layer current. So, combining the Maxwell equations, the surface plasmon dispersion relation in TM mode can be seen in Equation (6).k1 and k2 represent the longitudinal wave vectors of light up and below the interface. σ refers to the conductivity of graphene. According to the knowledge of surface plasmon wave, the real part of k1 and k2 must be greater than 0 (otherwise the surface plasmon waves do not exist), to ensure that the surface plasmon waves decrease from the interface to the medium internal. For the conductivity of graphene, the positive or negative values of the imaginary part in it will affect the positive and negative values of k1 and k2. Through this change, the excitation and cutoff of the surface plasmon waves in the TM mode can be controlled very easily.Among them, In the same way, the dispersion relationship of the graphene in TE mode can be obtained by Equation (7).From the Equation (7), the state of surface plasmon polariton waves can be controlled by changing the conductivity of graphene.To sum up these two parts, the changing of external voltage can directly influence the transmission or cutoff about surface plasmon polariton waves in this new structure. In this way, a new type of electro–optical switch modulator is proposed.According to This phenomenon is the opposite in TE mode. In the voltages of 0.5 V and 1 V, some incident wave bands can be spread in TE mode. But for the voltages in 1.5 V or 2 V, TE mode cannot be spread in the mid-infrared band .It can be observed from For the TM mode, all incident waves in Mid-infrared band can spread in 5 nm, which cannot be achieved in 10 nm, 15 nm, and 20 nm.2, BN, and Si.According to On the contrary, any Mid-infrared band waves can be spread in TM mode with Si. But the other materials only spread the incent waves in some mid-infrared bands.The two situations (1 V and 1.5 V) in According to Equation (7), when the external voltage is 1 V, the incident waves can spread in TE mode. It cannot spread in 1.5 V for the same mode. Therefore, it can be used as an electro–optical switch modulator.According to Equation (8), the bandwidth of the electro–optical modulator can be given as:R represents the resistance of the whole structure. C refers to the capacitance of the whole structure. After the calculation, the 3 dB bandwidth is about 34 GHz in this structure.Among them, pV represents the peak value of the external voltage. Using the 1 V and 1.5 V as the two switch voltages of this modulator, the energy loss is about 36.47 fJ/bit during one cycle.According to Equation (9), the energy loss of the electro–optical modulator can be given as:In this paper, a new electro–optical switch modulator based on surface plasmon polaritons of graphene was proposed. It used the tunable of graphene and the properties about surface plasmon polaritons to achieve the control of external voltage to the transmission of incident waves. This control way can be applied to design for an electro–optical switch modulator, which has 100% modulation depth, in theory. It cannot be obtained by traditional electro–optical modulator. In addition, it has very low energy loss (~36.47 fJ/bit), which is very adapt to products in engineering.


Antibiotic use during adolescence may result in dysbiosis-induced neuronal vulnerability both in the enteric nervous system (ENS) and central nervous system (CNS) contributing to the onset of chronic gastrointestinal disorders, such as irritable bowel syndrome (IBS), showing significant psychiatric comorbidity. Intestinal microbiota alterations during adolescence influence the expression of molecular factors involved in neuronal development in both the ENS and CNS. In this study, we have evaluated the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TrkB) in juvenile mice ENS and CNS, after a 2-week antibiotic (ABX) treatment. In both mucosa and mucosa-deprived whole-wall small intestine segments of ABX-treated animals, BDNF and TrKB mRNA and protein levels significantly increased. In longitudinal muscle-myenteric plexus preparations of ABX-treated mice the percentage of myenteric neurons staining for BDNF and TrkB was significantly higher than in controls. After ABX treatment, a consistent population of BDNF- and TrkB-immunoreactive neurons costained with SP and CGRP, suggesting up-regulation of BDNF signaling in both motor and sensory myenteric neurons. BDNF and TrkB protein levels were downregulated in the hippocampus and remained unchanged in the prefrontal cortex of ABX-treated animals. Immunostaining for BDNF and TrkB decreased in the hippocampus CA3 and dentate gyrus subregions, respectively, and remained unchanged in the prefrontal cortex. These data suggest that dysbiosis differentially influences the expression of BDNF-TrkB in the juvenile mice ENS and CNS. Such changes may potentially contribute later to the development of functional gut disorders, such as IBS, showing psychiatric comorbidity. In both CTR and ABX-treated whole-mount preparations BDNF-IR myenteric neurons were also immunoreactive to CGRP . CGRP immunoreactivity was also found in dense varicose fibres surrounding myenteric neurons . The percentage of BDNF-IR/CGRP-IR myenteric neurons in CTR preparations was 8.33±4.95% (n = 15) and significantly increased in preparations obtained from ABX-treated animals In control small intestine LMMP whole-mount preparations, BDNF antibody faintly stained the cytoplasm of few myenteric neurons  , panel G panels A and D, respectively. In mouse small intestine preparations, western blot analysis of TrkB revealed a band at 145 kDa. In ABX-treated mice, TrkB protein levels significantly increased both in the mucosa (p<0.05) and in mucosa-deprived whole intestinal wall (p<0.05) . In both layers, the levels of TrkB phosphorylated on Tyr816 significantly increased (p<0.05) .TrkB mRNA levels in mucosa and mucosa-deprived whole intestinal wall specimens significantly increased  after antibiotic treatment as reported in Fig 5, panel G). As observed after co-staining with the pan neuronal marker, HuC/D, TrkB antibody stained the soma of large and medium-sized neurons with prevalently an ovoidal shape. In myenteric ganglia, TrkB immunoreactivity was also found in prolongations discontinuously surrounding neurons, in interconnecting strands between ganglia and in bundles of fibres running along the longitudinal smooth muscle . After antibiotic treatment, a significant . In both CTR and ABX-treated whole-mount preparations TrkB-IR myenteric neurons were also immunoreactive to CGRP . CGRP immunoreactivity was found in the soma and nucleus of TrkB-IR neurons as well as in varicose fibres surrounding TrkB-IR neurons . In CTR preparations, 16.67±5.69% (n = 16) of TrkB-IR myenteric neurons was immunoreactive to CGRP. This percentage significantly increased in preparations obtained from ABX-treated animals .In control small intestine LMMP whole-mount preparations, TrkB antibody stained few neurons  . In contrast, both BDNF and TrkB protein levels were significantly lower in the hippocampus of ABX-treated mice (p<0.05 for both proteins) than in CTR . To investigate the pattern of distribution of BDNF and TrkB, we performed fluorescent immunohistochemical staining for both proteins in hippocampal slices of CTR and ABX-treated mice. In the hippocampus of both CTR and ABX-treated animals, BDNF and TrkB stained the CA1-CA3 and dentate gyrus (DG) subregions. However, after ABX treatment, BDNF staining decreased in the CA3 subregion , while TrkB staining was reduced in the DG subregion .In the hippocampus of ABX-treated mice, BDNF and TrkB mRNA levels were unchanged  with respect to CTR . BDNF and TrkB protein levels were also unchanged in the prefrontal cortex of ABX-treated mice  . Immunostaining for both BDNF and TrkB in the prefrontal cortex of ABX-treated mice did not display significant differences with respect to that observed in CTR animals .In the prefrontal cortex of ABX-treated animals, BDNF and TrkB mRNA levels remained unchanged  with respect to values obtained in CTR mice  mice [From a functional view point, BDNF exerts a facilitatory modulation on both motor and sensory gut functions via TrkB activation ,49. Admi/-) mice . The exc/-) mice ,45. This/-) mice . Accordi/-) mice . In our /-) mice , such as/-) mice ,53. Sinc/-) mice , we cann+/- mice developed lower colonic nociception under both control conditions and after trinitrobenzesulfonic acid-induced colitis [As a neurotransmitter/neuromodulator, BDNF released from dorsal root ganglia plays a fundamental role in the modulation of chronic pain conditions caused by peripheral inflammatory responses . Recentl colitis . Enhance colitis . Co-loca colitis , indicat colitis ,57.Overall, the changes observed in enteric BDNF-TrkB signaling after antibiotic-induced microflora depletion in the juvenile mouse small intestine share many similarities with the alterations of BDNF signaling, previously observed in animal models of IBS and in IBS patients. IBS is a functional GI disease, displaying comorbidity with psychiatric disorders and is associated with anxiety and mood disorders . In thisIn summary, the present data suggest that a dysbiosis induced by a massive antibiotic treatment during adolescence may alter BDNF and TrkB expression both in the ENS and CNS, although with different outcomes in the two nervous systems, later in life. We cannot exclude that these molecular changes may contribute to alter specific neurotransmitter pathways, involved in the development of functional gut diseases, such as IBS. These results further strengthen the concept that appropriate manipulation of the gut microbiome during adolescence, may reduce the probability of developing severe disorders that could affect the gut and the CNS later. This is all the more interesting in view of the worldwide increasing incidence of functional gastrointestinal disorders affecting the adolescent and young population ,65.


ALys124. The unbinding process prediction showed that the PD-L1 dimerization kept stable upon the dissociation of ligands. It's indicated that the formation and stability of the small-molecule inducing PD-L1 dimerization was the key factor for the inhibitory activities of these ligands. The contact analysis, R-group based quantitative structure-activity relationship (QSAR) analysis and molecular docking further suggested that each attachment point on the core scaffold of ligands had a specific preference for pharmacophore elements when improving the inhibitory activities by structural modifications. Taken together, the results in this study could guide the structural optimization and the further discovery of novel small-molecule inhibitors targeting PD-L1.Recently, small-molecule compounds have been reported to block the PD-1/PD-L1 interaction by inducing the dimerization of PD-L1. All these inhibitors had a common scaffold and interacted with the cavity formed by two PD-L1 monomers. This special interactive mode provided clues for the structure-based drug design, however, also showed limitations for the discovery of small-molecule inhibitors with new scaffolds. In this study, we revealed the structure-activity relationship of the current small-molecule inhibitors targeting dimerization of PD-L1 by predicting their binding and unbinding mechanism via conventional molecular dynamics and metadynamics simulation. During the binding process, the representative inhibitors (BMS-8 and BMS-1166) tended to have a more stable binding mode with one PD-L1 monomer than the other and the small-molecule inducing PD-L1 dimerization was further stabilized by the non-polar interaction of Ile54, Tyr56, Met115, Ala121, and Tyr123 on both monomers and the water bridges involved in  The blockage of the protein-protein interaction (PPI) between programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) can reactivate the effector functions of T cell and eliminate tumor phenotypes with significant PD-L1 expression  company, the compounds with (2-methyl-3-biphenylyl)methanol scaffold were privileged for inducing the dimerization of PD-L1 and interacted with the hydrophobic tunnel formed by two PD-L1 monomers  with known inhibitory activities and complex crystal structures were selected to perform molecular dynamics simulations. During the formation process, both monomer and dimer systems of PD-L1 in complex with small-molecule ligands were applied to evaluate the stabilities of binding modes between ligands and PD-L1. The binding free energy calculation by MM-PBSA and MM-GBSA  and small-molecule ligands were derived from the PDB database with accession number of For dimer systems of BMS-8 and BMS1166, two PD-L1 monomers were selected as the receptor, while small-molecule inhibitors were selected as the ligand. Both MM-PBSA and MM-GBSA methods were performed to calculate the binding free energy of BMS inhibitors according to the equation below:Where < > represents the average value for all the snapshots used for MM-PBSA and MM-GBSA calculation. Different energy terms can be estimated as follows:TΔS) was calculated by 100 snapshots from the last 20 ns trajectory.500 snapshots were extracted from the last 20 ns trajectories and used for MM-PBSA and MM-GBSA calculation. The parameter settings during MM-PBSA and MM-GBSA calculation were referred to the previous works published by our group  to equilibrate the systems with restraining force constant of 5 kcal mol−1·Å−2 and all restraints released therewith. Finally, the equilibrated structures restarted from the NVT simulation were used for metadynamics simulations.Metadynamics simulations have been widely used to predict the unbinding pathways and dissociation energy barrier of ligands for ligand-target systems  during time span (kτ). In this study, the deposition time (τ) was set as 1 ps to give enough dissociation time for ligands without adding biasing potential on the dissociation boundary. The Gaussian width (σ) of CV1 and CV2 were set to 0.8 Å and 0.02 rad, respectively. As for the well-tempered metadynamics, the height of the Gaussian potential (W) is affected by a parameter ΔT as Equation (8). The initial hill height (W0) of Gaussian potential was set to 0.6 kcal/mol·ps and the bias-factor (γ) was set to 10 with a temperature (T) of 300 K to control the decrease rate of the biasing potential as Equation (9).Metadynamics could reconstruct the free-energy surface as a function of specific collective variables (CVs). The general formalism of history-dependent Gaussian potential was shown as Equation (7). 50) was analyzed and the importance of six key pharmacophore elements including hydrogen bond acceptor (A), hydrogen bond donor (D), hydrophobic group (H), negative ionic group (N), positive ionic group (P), and aromatic ring (A) were evaluated at each attachment point. During the process, the error and the importance were both set as 0.30. Eight representative small-molecule inhibitors  with substitutions on R1, R2, or R3 were selected to perform molecular docking to further study the binding modes. In order the compare the effect of R-groups, the core scaffold atoms with SMILES of “cOCc(c1C)cccc1c” were constrained with RMSD of 0.5 angstrom while other atoms were selected flexible. The standard precision (SP) docking score was used to evaluate the binding poses. The molecular docking was performed in Schrödinger 2015 software suite .The pharma R-group quantitative structure-activity relationship (RQSAR) models tended to be an effective approach for the SAR evaluation of the congeneric series of compounds  and a worse entropy contribution (–TΔΔS) than BMS-8, which were consistent with the stability difference of BMS-8 and BMS-1166 dimer complex.The conformational stabilities of the dimer systems were evaluated by root mean square deviation (RMSD) and mean square root fluctuation (RMSF) as shown in ATyr56, AMet115, AAla121, ATyr123 and BIle54, BTyr56, BGln66, BMet115, BAla121 as shown in AIle54, ATyr56, AMet115, AAla121, AAsp122, ATyr123, AArg125 and BIle54, BTyr56, BVal76, BMet115, BAla121, BAsp122 as shown in BGln66 with an occupancy of 57.21%, while the BMS-1166 dimer system also formed hydrogen bond between the ammonium group on BMS-1166 and the carboxyl group of AAsp122. The binding mode analysis of substitute groups on BMS-8 and BMS-1166 with the interfacial residues on PD-L1 indicated that the interaction with the peripheral residues including AIle54, AArg125, BVal76, and BAsp122 could significantly enhance the inhibitory activities of BMS small-molecule inhibitors.The key residues on two PD-L1 monomers interacting with ligands were recognized by per-residue energy decomposition. The energy contribution for each residue were decomposed into the sidechain part and the backbone part, the non-polar part and the polar part as shown in AAsp122, ATyr123, ALys124, and BGln66 were stable in both dimer systems. Both ligands formed a strong water bridge with ALys124 with an occupancy higher than 90%, which indicated that ALys124 had a significant effect on the stabilities of the ligand conformations.In order to analyze the effect of solvent on PD-L1 dimer complex, water occupancies and water bridges involved in receptor-ligand interaction were both calculated. As shown in The free energy landscape of the unbinding processes of both BMS small-molecule inhibitors were constructed by CV1 and CV2. The distribution of minima in the landscapes showed that the most stable conformational state in the unbinding process was corresponding to the conformational states of the initial crystal structures as shown in ATyr56 and got away from the pocket formed by PD-L1 dimer. In order to monitor the conformational change of the pocket formed by PD-L1 monomers, the distance between chain A and chain B were calculated by the distance between Ile54, Tyr56, Met115, Ala121, Tyr123 on each chain as shown in From the free energy estimation of different conformational states, it can be seen that the conformational states of the crystal structures were much more stable than the other transition conformational states along the unbinding process. Therefore, the dissociation of small-molecule ligands of the initial conformational states tended to be the most important intermediate process for the unbinding of small-molecule ligands, which were corresponding to the minima of CV1 at 12.54 Å in BMS-8 dimer systems and the minima of CV1 at 10.95 Å in BMS-202 dimer systems. The corresponding transition states were extracted from the trajectories as shown in Taken together, the most possible deduction for the interaction mechanism of BMS small-molecule inhibitors with PD-L1 was depicted as shown in 50 ranging from 9.492 μM to 1.4 nM. As shown in 50 and the experimental pIC50 was 0.7729. According to the evaluation of six key pharmacophore elements in AIle54, ATyr56, BAsp122, BTyr123. The hydrogen bond acceptor and hydrophobic groups at R2 were favorable for BMS inhibitors such as the 2, 3-dihydro-1, 4-benzodioxinyl group on BMS-114, BMS-200, BMS-1001, and BMS-1166. The analysis of effect of solvent in dimer systems showed that the substituent groups at R4, R5, R6, and R7 were exposed to solvent environment. The hydrophobic groups at R4 were favorable for BMS inhibitors, which corresponded to the fact that the bromine atom on BMS-8 and the chlorine atom on BMS-1166 had a close contact with BIle54. The hydrophobic group at R6 was adverse while the negative ionic group was favorable. The substituent group at R6 of BMS-8 mainly interacted with BTyr56 and BGln66, however, that of BMS-1166 mainly interacted with AThr20 and AAsp122. Nevertheless, the substituent group at R6 of BMS-8 and BMS-1166 both formed hydrogen bonding with PD-L1. The positive ionic at R7 was adverse while the hydrogen bond acceptor and aromatic ring were favorable. The substituent group at R7 of BMS-1166 formed interaction with AAsp122, ATyr123, ALys124, AArg125, BTyr56, and BGln63. The comparison of the contact residues between BMS-8 and BMS-1166 indicated that the substituent group at R2 and R7 strongly strengthened the interactions with the conformation A of PD-L1, which was consistent with the stability of the monomer complex of conformation A and BMS-1166.110 BMS small-molecule inhibitors with 2-methyl-3-(phenoxymethyl)-1,1′-biphenyl scaffolds were tested with diverse inhibitory activities with ICBAsp122 (interacting with R1 to R3) and AAsp122, ALys124, BTyr56, BGln66 (interacting with R4 to R7). The binding mode analysis of novel series of triazolopyridines designed by Qin et al. also revealed the retaining hydrophobic interaction with Tyr56, Met115, and Ala121 on both chain of PD-L1 and extra π-π stacking with the BTyr56 and π-anion interactions with AAsp122 (Qin et al., The further molecular docking study of eight representative small-molecule inhibitors showed that the docking scores had a good linear correlation with the experimental inhibitory activities . The furIn this study, we used multiple molecular modeling methods to study the detailed molecular mechanism of the interaction between BMS small-molecule inhibitors and PD-L1. A detailed mechanism of the interaction process between small-molecule inhibitors and PD-L1 was proposed and validated by molecular dynamics simulations.ALys124. The unbinding pathway prediction also indicated that the tunnel formed by PD-L1 dimers tended to be stable upon the getting away of BMS-inhibitors. The R-group QSAR model suggested that the substituents at R2, R4, R6, and R7 had a significant effect on the inhibition activities of BMS inhibitors. The structural modification with these substituent positions tended to be an effective way to improve the inhibition activities of BMS inhibitors. Taken together, this study would provide a comprehensive view of the inhibition mechanism for BMS small-molecule inhibitors and guide the further development of more potential small-molecule inhibitors targeting PD-L1.The BMS small-molecule inhibitors tended to interact with one PD-L1 monomer first and further formed dimer with the other monomer for an advantage of stability. The results of binding free energy and water occupancy calculation revealed the key stability factors for ligand-induced PD-L1 dimers including the hydrophobic contribution of Ile54, Tyr56, Met115, Ala121, and Tyr123 on both monomers and the water bridges involved in The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.DS, HL, and XY designed the research. DS and XY were responsible for the writing and revising of the manuscript. XA, QB, SZ, and ZB were responsible the main data analysis in the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.