Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) share overlapping genetic causes and disease symptoms, and are linked neuropathologically by the RNA binding protein TDP-43 (TAR DNA binding protein-43 kDa). TDP-43 regulates RNA metabolism, trafficking, and localization of thousands of target genes. However, the cellular and molecular mechanisms by which dysfunction of TDP-43 contributes to disease pathogenesis and progression remain unclear. Severe changes in the structure of neuronal dendritic arbors disrupt proper circuit connectivity, which in turn could contribute to neurodegenerative disease. Although aberrant dendritic morphology has been reported in non-TDP-43 mouse models of ALS and in human ALS patients, this phenotype is largely unexplored with regards to TDP-43. Here we have employed a primary rodent neuronal culture model to study the cellular effects of TDP-43 dysfunction in hippocampal and cortical neurons. We show that manipulation of TDP-43 expression levels causes significant defects in dendritic branching and outgrowth, without an immediate effect on cell viability. The effect on dendritic morphology is dependent on the RNA-binding ability of TDP-43. Thus, this model system will be useful in identifying pathways downstream of TDP-43 that mediate dendritic arborization, which may provide potential new avenues for therapeutic intervention in ALS/FTD. Mutations in the TARDBP gene which encodes TAR DNA-binding protein of 43 kDa (TDP-43) have been linked to both ALS and FTD in humans6. The connection between ALS and FTD has been further confirmed at the molecular level by the identification of TDP-43 as the major component of ubiquitin–positive inclusions in both ALS and the most common pathological form of FTD8.Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are clinically linked, rapidly progressing neurodegenerative diseases that affect both distinct and overlapping regions of the brain. Nearly 50% of ALS patients show some level of cognitive impairment while 25–30% of FTD patients exhibit motor neuron dysfunction12, several lines of evidence suggest that pathologically altered TDP-43 causes neurodegeneration by both GOF and loss-of-function (LOF) mechanisms. First, in patients, cytoplasmic TDP-43 inclusions occur concomitantly with depletion of TDP-43 protein from the nucleus8. Second, TDP-43 has been shown to downregulate the expression of its own mRNA transcript13, and in transgenic mice, overexpression of human TDP-43 results in downregulation of endogenous TDP-4314, suggesting an impairment of TDP-43 function by an interplay between both LOF and GOF mechanisms. Third, TDP-43 LOF in animal and cellular models phenocopies many defects seen when overexpressing either wild-type or mutant forms of TDP-43 that are implicated in human disease17. Thus, LOF and GOF hypotheses are not mutually exclusive and it is possible that both mechanisms contribute to neurodegeneration18.While aggregate-forming proteins are often thought to cause disease via toxic gain-of-function (GOF) mechanisms20, as well as miRNA processing25 and mRNA splicing32. Although it mostly resides in the nucleus, TDP-43 also shuttles to the cytoplasm33 where it is involved in stress granule formation40, axonal transport of target mRNAs42, and mRNA translation47. TDP-43 interacts with several proteins that are implicated in its RNA-processing functions48 including the heterogenous nuclear ribonucleoprotein complex which binds to the TDP-43 carboxy-terminal domain49. Despite the numerous, well-described functions and interactions of TDP-43, it is not well understood exactly which TDP-43-dependent cellular processes become defective in ALS/FTD and contribute to disease etiology.TDP-43 is a highly conserved, ubiquitously expressed, multifunctional nucleic acid-binding protein composed of two RNA recognition motifs (RRM), nuclear localization (NLS) and export signals (NES), and a carboxy-terminal glycine rich region. TDP-43 regulates transcription52. In animal models of ALS, cortical neuronal degeneration along with regression of apical dendrites has been observed in presymptomatic SOD1G39A mice54. Cortico-spinal Motor Neurons  also show apical dendrite regression in sporadic, familial, and FTD-ALS patients, indicating the loss of the dendritic arbor as an important marker of cellular pathology in ALS55. Given that TDP-43 is involved in the splicing, transport, and stability of several mRNAs which encode proteins that play important roles in neuronal arbor elaboration and synaptic function57, we asked whether TDP-43 dysfunction altered neuronal morphology.Defects in dendritic arbor elaboration and maintenance have been associated with many neurodegenerative diseasesTo explore this question, we took the approach of overexpressing or knocking down TDP-43 in cultured mammalian neurons isolated from rat hippocampus or cortex and asked if manipulating TDP-43 protein levels interfered with dendritic complexity. Our results demonstrate that both overexpression and knockdown of TDP-43 result in reduced dendritic branching, without affecting neuronal viability. In addition, the effect of TDP-43 overexpression on dendritic loss is dependent on TDP-43 RNA binding. Our findings demonstrate that aberrant levels of TDP-43 disrupt neuronal morphology, an observation that has important implications for understanding disease pathology.62. Our first experiments were performed using cultured prenatal rodent hippocampal neurons. We chose this primary neuronal culture system since it has been used extensively to investigate the cell biological mechanisms of dendritic growth and branching67 and because of its established use as a model to study TDP-43 biology69. We isolated hippocampal neurons from E18 rat pups and cultured them on a glial monolayer. Neurons were co-transfected at 2 days in vitro (DIV) with a plasmid expressing GFP to visualize neuronal morphology, and either a plasmid expressing the human TDP-43 gene under the control of a strong promoter (pCMV) at a concentration of 200 ng/well or 500 ng/well in a 24-well plate, or an empty vector control at 500 ng/well . Neurons were co-transfected with GFP and either an empty vector control (500 ng/well), TDP-43 (500 ng/well), or FUS (500 ng/well). We analyzed dendritic complexity of the transfected neurons using Sholl analysis and found that FUS overexpression did not significantly alter dendritic branching compared to controls  is an RNA-binding protein that is genetically linked to ALS and FTD and is structurally and functionally similar to TDP-43To determine if reduced dendritic complexity could be attributed to poor cell health induced by TDP-43 overexpression, we asked if cortical neurons overexpressing TDP-43 displayed increased cell death relative to control transfected neurons. We assayed cell death using propidium iodide (PI), a membrane-impermeable nucleic acid stain that labels nuclei of dead cells. We quantified the percentage of transfected cells (co-labeled with GFP) that were positive for PI at DIV 4 or DIV 7 in control or TDP-43 overexpressing neurons that were transfected at DIV 2. We found no significant difference in the percentage of PI-positive neurons between the two conditions , which promotes microtubule stability in dendrites. We measured the levels of MAP2 in cortical neurons after overexpression of TDP-43 at 500 ng/well , or an empty vector control. On DIV 7, we fixed these neurons, and confirmed depletion of TDP-43 by immunostaining with a polyclonal antibody recognizing TDP-43  or TDP-43 (500 ng/well) over the time-course of our experiment, using a polyclonal TDP-43 antibody that recognizes both rat and human TDP-43, starting at 2 days post-transfection. Relative levels of TDP-43 expression were calculated by normalizing intensity of TDP-43 staining within the nucleus and cytoplasm to intensity of staining with a control antibody labeling TFIIS80. At DIV 4 (2 days post-transfection), we observed that neurons transfected with the TDP-43-expressing plasmid displayed approximately a 4-fold increase in TDP-43 protein levels compared to controls  or a TDP-43-expressing plasmid (2 µg/dish). These vector concentrations scale proportionally with our previous transfections on 24-well plates at 500ng/well. The dendrites from each image were traced and measured for total dendrite length at each time point. We observed that dendrites from control neurons increased in length at each time point over the 40-hour imaging window Fig. . HoweverSimilarly, hippocampal neurons overexpressing TDP-43 showed a consistent reduction in neurite outgrowth by Sholl analysis over time. We transfected hippocampal neurons with either a control plasmid (500 ng/well) or a TDP-43 plasmid (500 ng/well) and then fixed and imaged a subset of these neurons on each successive day starting at 2 days post-transfection (DIV4). We found that TDP-43 overexpressing neurons did not exhibit a statistically significant difference in dendritic complexity at DIV 4 (48 hours after transfection) Fig. . HoweverTARDBP) have been identified in both familial and sporadic ALS patients83. Most of these mutations reside in the C-terminal glycine rich domain, which is essential in mediating protein-protein interactions49. A few of these mutations have been shown to impair the trafficking of TDP-43-containing RNP granules in axons41. In addition, mutant forms of TDP-43 have been reported to increase TDP-43 cytoplasmic localization in cultured cortical neurons68. Thus, we hypothesized that TDP-43 mutations might exacerbate the decreased dendritic branching phenotype observed upon overexpression of wild-type (WT) TDP-43. To test this hypothesis, we generated two TDP-43 constructs, each carrying a single point mutation previously described in human patients: TDP-43 A315T and TDP-43 M337V 33 and it has been shown to be enriched in the cytoplasmic compartment90. Furthermore, TDP-43 CTF also lacks its primary RNA-recognition motif (RRM1), so it cannot interact with its RNA targets26. We sought to determine if overexpression of the C-terminal domain could also suppress dendritic branching. To address this question, we co-transfected cultured cortical neurons on DIV 2 with a GFP-expressing plasmid along with an empty vector (500 ng/well), or TDP-43 (500 ng/well), or a plasmid expressing a truncated TDP-43 (500 ng/well) which only expresses the C-terminal region of TDP-43 composed of amino acid residues 170–414  TF) Fig. . These nTF) Fig. . These r94 and enhances splicing activity94. Therefore, we investigated whether the overexpression of a TDP-43 construct containing amino acid residues 10–414 (hereafter referred to as TDP-43 ∆N) was sufficient to suppress dendritic branching. Cultured cortical neurons were co-transfected on DIV 2 with a GFP-expressing plasmid along with an empty vector (500 ng/well), or TDP-43 (500 ng/well), or TDP-43 ∆N (500 ng/well). On DIV 7, these neurons were fixed and assayed by Sholl analysis. We found that TDP-43 ∆N overexpression had no effect on dendritic branching 26. We co-transfected cortical neurons with a GFP-expressing plasmid along with an empty vector (500 ng/well), TDP-43 (500 ng/well), or a plasmid expressing TDP-43 5FL (500 ng/well) on DIV 2 and fixed these neurons on DIV 7. Overexpression of TDP-43 5FL had no effect on dendritic complexity  were a gift from Dr. Zuoshang Xu 90. Plasmids expressing full-length human FUS (Addgene plasmid # 29609) and TDP-43 5FL (Addgene plasmid # 84914) was a gift from Aaron Gitler . pCSCMV:tdTomato was a gift from Gerhart Ryffel (Addgene plasmid # 30530). Full-length TDP-43, FUS, and TDP-43 5FL were cloned into pCMV-myc vector backbone using Gibson Assembly (NEB). Constructs containing various mutations of TDP-43 were generated from pCMV-myc hTDP-43 plasmid by site-directed mutagenesis using QuickChange Site Directed Mutagenesis Kit . TDP-43 ∆N was generated by cloning from the pCMV-myc hTDP-43 plasmid. Primers used to generate indicated mutations are as follows:Plasmids expressing full length human TDP-43 (Addgene plasmid # 28206) and C-terminal truncated human TDP-43  and laminin (3.4 μg/ml) in 24 well plates. Dissociated hippocampal or cortical neurons from E18 rat embryos were plated on this layer of astrocytes at the density of 80,000 cells/well, and grown in Neurobasal medium supplemented with B27 (Thermo Fisher) at 37 °C.Dissociated rat cortical or hippocampal neurons were cultured on a feeder layer of astrocytes as described previously114, which yields a low transfection efficiency, allowing development of transfected neurons within a large population of unaffected neurons, as well as better visualization of neuronal morphology for individual transfected neurons. All experimental conditions were co-transfected with pCMV-GFP plasmid at 500 ng/well to visualize cellular morphology. In addition, all wells were co-transfected with either pCMV-myc plasmid for control empty-vector conditions, or other plasmid DNA as described.Neurons were transfected at the desired stage by using the calcium phosphate transfection methodTransfected neurons were fixed at the desired time-point using 4% paraformaldehyde + 4% sucrose solution in PBS for 8 minutes at room temperature followed by 3 washes in 1X PBS. Images of individual transfected neurons were acquired on Olympus Fluoview 300 confocal microscope using a 20X oil objective  in order to image the entire dendritic arbor . All conditions were imaged at similar acquisition settings for laser power and detector gain in a blinded manner, and unblinded only after analysis.Maximum Intensity Projections (MIP) were generated from individual Z stacks using ImageJ. For Sholl analysis, a series of 11 concentric circles  centered at the soma, starting at 10 µm, was overlaid on the MIP using the ‘Concentric circles’ plugin. The number of dendrites crossing each circle was measured manually. For each experimental condition, a total of 15–30 neurons were analyzed from 2–3 coverslips with 5–15 neurons per covers lip. The reported results combine data from at least 2 independent biological replicates. For measuring total neurite length and branch tip number, neurites were traced using the NeuronJ plugin in ImageJ. The sum length of all the neurites was calculated for 10–15 neurons from 2–3 coverslips and the results were combined from 2 independent biological replicates.To measure TDP-43 protein levels, sum projections were generated from individual Z stacks with ImageJ. In ImageJ, using GFP as a marker for transfected cells, a region of interest was drawn around the soma and fluorescence intensity was measured in that area in the TDP-43 channel. TFIIS fluorescence intensity was measured by drawing a region of interest around the Hoechst-33342-labeled nucleus, and fluorescence intensity was measured in the TFIIS channel. TDP-43 intensity was normalized to TFIIS intensity for every cell.Nuclear vs cytoplasmic levels of TDP-43 staining were measured as follows. For nuclear TDP-43 levels, the region of interest was defined by the Hoechst-33342 labeling and TDP-43 intensity was measured in the TDP-43 immunostained channel. The TDP-43 levels in the cytoplasm were determined by drawing a region of interest around the GFP-filled soma, measuring TDP-43 intensity in the TDP-43 channel, and subtracting the previously measured nuclear TDP-43 intensity.MAP2 levels were quantified by measuring fluorescence intensity of MAP2 immunostaining from empty vector or TDP-43-transfected cells (500 ng/well). Sum projections of each condition were generated using ImageJ, with GFP as a marker for transfected cells. Average intensity was calculated by taking the mean intensity from 2 separate dendrites of a transfected neuron followed by normalizing to the average intensity from 2 other dendrites from non-transfected cells.2, 0.814 mM MgSO4, 1 mM NaH2PO4, 20 mM HEPES, 50 mM D-glucose, 0.1% BSA, 320 mOsm, pH 7.2) for 1 hour at 37 °C followed by 1X PBS wash. Cultures were fixed with 4% PFA + 4% sucrose solution in PBS. GFP-positive neurons were counted manually using a 60x objective and an epiflourescence microscope, and the presence or absence of PI fluorescence was noted for each GFP-positive neuron by flipping between filter sets. For each experimental condition, 2 coverslips each from 2 independent experiments were analyzed.Hippocampal neurons were cultured in 24-well plates as described and transfected with pCMV-GFP plasmid along with either empty pCMV-myc control plasmid at 500ng/well or full-length hTDP-43 at 500 ng/well. Neurons were incubated in 2 µg/ml Propidium Iodide solution (Life Technology) in HEPES buffer  from ProteinTech (used at 1:500); α-TFIIS (611204) from BD Transduction Laboratories (used at 1:275); α-MAP2 (M4403) from Sigma-Aldrich (used at 1:1000). Secondary antibodies were conjugated to Cy-3 or Cy-5 . Coverslips containing transfected neurons were fixed as described above followed by 3 washes with 1X PBS. The coverslips were then incubated with primary antibodies) diluted in gelatin blocking buffer at 4 °C overnight in a humidified chamber (30 minutes at room temperature for MAP2). Coverslips were then washed 3 times with 1X PBS and incubated with secondary antibodies at room temperature for 2 hours. Coverslips were then washed 3 times with 1X PBS, submerged in MilliQ water and then mounted on glass microscope slides with Aquamount (Lerner Laboratories). Neurons were imaged using an Ni-E inverted microscope equipped with a Nikon C2 confocal head and a 60X oil (NA 1.4) objective, using similar acquisition settings for laser power, offset and detector gain across conditions in a blinded manner.An astrocyte feeder layer and cortical neurons were plated as described above on 35 mm glass-bottom dishes. 4 hours after plating, neurons were transfected via the calcium phosphate method with a plasmid expressing TdTomato to fill the cell and an empty vector control or a vector expressing TDP-43; our empirical observation was that TdTomato is less phototoxic than GFP for these live imaging experiments. The next day (~24 hours later) the neurons were placed in an OKO labs temperature controlled environmental chamber  and 20–30 neurons were randomly selected from each condition. Images of the selected neurons were acquired every 8 hours for 40 hours using an Ni-E inverted microscope equipped with a Nikon C2 confocal head and a 40X (NA 0.95) objective. Images were collected using Nikon Elements AR software. The experimenters were not blinded to the condition during the selection of neurons but were blinded to each condition during analysis. Images from each time point were analyzed by tracing individual dendrites using ImageJ.Supplementary Information


Novel hybrid metal-graphene metamaterials featuring dynamically controllable single, double and multiple plasmon induced transparency (PIT) windows are numerically explored in the terahertz (THz) regime. The designed plasmonic metamaterials composed of a strip and a ring with graphene integration generate a novel PIT window. Once the ring is divided into pairs of asymmetrical arcs, double PIT windows both with the spectral contrast ratio 100% are obtained, where one originates from the destructive interference between bright-dark modes, and the other is based on the interaction of bright-bright modes. Just because the double PIT windows are induced by two different mechanisms, the continuously controllable conductivity and damping of graphene are employed to appropriately interpret the high tunability in double transparency peaks at the resonant frequency, respectively. Moreover, multiple PIT windows can be achieved by introducing an additional bright mode to form the other bright-bright modes coupling. At the PIT transparent windows, the dispersions undergo tremendous modifications and the group delays reach up to 43 ps, 22 ps, and 25 ps, correspondingly. Our results suggest the existence of strong interaction between the monolayer graphene layer and metal-based resonant plasmonic metamaterials, which may hold widely applications in filters, modulators, switching, sensors and optical buffers. However, rigorous conditions for the realization of conventional EIT effect have significantly limited the practical applications. Similar to the EIT effect, plasmon induced transparency (PIT), which is strong interaction among the bright-dark modes5 or bright-bright modes9, has been proposed in various plasmonic platforms such as plasmonic devices11, planar metamaterials14 and large area hybrid plasmon-waveguide systems15. Among different plasmonic systems, planar metamaterials become the research hot spot for the negligible propagation loss.Electromagnetically induced transparency (EIT) effect, which is produced by the quantum destructive interference between the two excitation pathways in laser-activated multiple level atomic systems16, tight field confinement18, and the gate-voltage dependent feature21. Therefore, an effective route for control and manipulation of graphene-based dynamically controllable PIT effect has been proposed and the majority of these researches focus on the shifting of the whole PIT window accompanied by changing the Fermi energy of graphene25. For some applications, such as frequency-selective filter, it may be highly desirable to control specific transmission intensity while keeping the resonant frequency fixed. Recently, there have been many efforts in studying tunable metamaterials based on hybrid metal-graphene. Zhang et al. designed independently tunable perfect absorber based on hybrid metal-graphene metamaterials at mid-infrared frequencies26 and Sun et al. proposed independently tunable dual-band PIT based on hybrid metal-graphene25. Both of them extensively investigated the shift of the resonant frequency by changing the Fermi energy of the graphene. However, the intensity modulation of PIT has been rarely reported and the key role of graphene in the hybrid metal-graphene metamaterials has not been fully understood. Hence, the intensity modulation of PIT and how graphene works remain to be studied in the hybrid metal-graphene metamaterials.Graphene has been regarded as a new class of promising plasmonic material in photonics and optoelectronics due to many novel optical properties. At mid-infrared frequencies and terahertz (THz) frequencies, graphene has manifested a lot of unprecedented advantages including low propagation lossMotivated by the previous observations, we investigate the dynamically controllable single, double and multiple PIT effects based on hybrid metal-graphene metamaterials by varying the Fermi energy in graphene, which consist of a strip and a ring with graphene integration structure. Both the strip and the ring are served as bright modes, which are induced by electric dipole oscillations. A novel PIT window caused by the hybridization of bright-bright modes can be controlled because of the oscillations of the metallic constructions damped by the dissipation of the graphene layer. By dividing ring into pairs of asymmetrical arcs, the other PIT transparency window with the spectral contrast ratio 100% emerges due to the excitation of magnetic dipole resonance. The continuously controllable conductivity of graphene can also explain the dynamically controllable amplitude of this PIT. Then, by introducing an additional bright mode to form the other bright-bright modes coupling, multiple PIT windows can be achieved and can be effectively controlled with different modulation depth approximately 39%, 86%, and 89% when the Fermi energy of graphene increases from 0.0 eV to 0.8 eV. At the transparent windows, the tremendous changes in the dispersions and the group delays reach up to 43 ps, 22 ps, and 25 ps, correspondingly. Compared with previously PIT effects, our structured plasmonic metamaterials presented have high feasibility and easy modulation.27. Therefore, by applying the different gate voltages between the top electrode and the bottom electrode, we can achieve the flexibly controllable Fermi energy of graphene finger sets28. The unit cell is arranged in a two-dimensional array with a lattice constant Px = Py = 80 μm, which is illustrated in Fig. r = 18 μm with a ring width of 6 μm and spans different angles corresponding to θ′ = 155° and 180°. The strip placed 30 μm away from the center of the ASR has length L = 60 μm, width w = 6 μm. All metallic elements are chosen to be aluminum (Al) and have the same thickness t = 200 nm.Figure 30. In the ASR, electric dipole, performed as the bright mode, is accessible from the free-space excitation with a broad transmission dip caused by the strong radiative losses, while magnetic dipole, regarded as the dark mode, cannot be directly coupled to the incident field because the magnetic arm of the incident plane wave is perpendicular to the array plane. However, the magnetic dipole can interact through near-field coupling with the bright mode, resulting in collective resonances of the ASR and the suppression of radiation damping32, forming the spectrally narrow dark mode.To our knowledge, the ASR supports electric dipole with currents flow symmetrically and magnetic dipole with currents flow antisymmetricallyθ′. As shown in Fig. f1 = 0.945 THz, f2 = 0.962 THz, and f3 = 1.073 THz. From the Ez distributions at f1 = 0.945 THz  and with graphene at different Fermi levels are compared in Fig. 34:σg and Eres represent the conductivity of graphene and the resonant electric field, respectively. Due to the surface conductivity of graphene is relatively small, a big enough dissipative power density also demands that the resonant electric field is large enough. In the entire frequency range, only the frequency correspond to the PIT Peak has a large enough resonant electric field, thus, the transmission intensity of PIT peak undergoes strong modulation at the fixed frequency. When the resonant electric field is large enough at the resonant frequency, the enhancement of graphene conductivity via increasing its Fermi energy can strengthen the dissipative power density, as a result, the transmission intensity of PIT peak is substantially reduced and even turned off, which is in agreement with our trend presented above. The dynamically controllable PIT effect together with the physical mechanism can be strategically crucial in designing active hybrid metal-graphene metamaterials.To explore the physical mechanism behind this novel phenomenon, the surface charge density distributions at the resonant frequency without  with the graphene layer, another strip regarded as bright mode is implemented to validate the multiple dynamically controllable PIT effects. The unit cell is shown in the inset of Fig. Based on the previous structure comprising of a strip and ASR  method, using perfectly matched layer (PML) absorbing conditions in the propagation directions of the normally incidence plane wave and the periodical boundary conditions in the x and y directions to model the periodic array corresponding to the saturated array size. In the THz regime, the complex relative permittivity of aluminum 39:e, kB, T, ћ, Γ, Ef, and ω is the electron charge, the Boltzmann’s constant, the temperature, the reduced Planck’s constant, the phenomenological scattering rate, the Fermi energy of graphene, and the angular frequency, respectively. The relaxation time μ, the Fermi energy of graphene Ef, and the Fermi velocity vf.. Notably, a high relaxation time in the graphene also reduces the transmission attenuation and means the difficulty in fabrication. So, we select the relaxation time τ = 70 fs, which consistent with the refs41. Figure Compared with the noble metal, graphene’s surface conductivity can be flexibly tailored by applying a gate voltage. In our simulations, graphene is modeled as a 2D flat plane whose conductivity presented by the Kubo formula


Van der Waals (vdW) interaction between two-dimensional crystals (2D) can trap substances in high pressurized (of order 1 GPa) on nanobubbles. Increasing the adhesion between the 2D crystals further enhances the pressure and can lead to a phase transition of the trapped material. We found that the shape of the nanobubble can depend critically on the properties of the trapped substance. In the absence of any residual strain in the top 2D crystal, flat nanobubbles can be formed by trapped long hydrocarbons . For large nanobubbles with radius 130 nm, our atomic force microscopy measurements show nanobubbles filled with hydrocarbons (water) have a cylindrical symmetry (asymmetric) shape which is in good agreement with our molecular dynamics simulations. This study provides insights into the effects of the specific material and the vdW pressure on the microscopic details of graphene bubbles. Graphene nanobubbles can act as enclosures for holding small volumes of substances. Here the authors find a correlation between bubble shape and the encapsulated material providing a potential method for determining the graphene bubble content by its deformation. Recently, for large bubbles (R>50 nm), Khestanova et al.hmax/R where hmax is the maximum height and R is the base radius of the bubble filled with hydrocarbons, as measured by AFM10Using membrane theory, nonlinear plate theory, and the ideal gas model, Yue R=4 nm and height 0.5 nm, it was shown that this results in a pseudo magnetic field of 100 Tesla , the height, and radius of the bubble. For hmax∼1 nm this results into Phyd ≈1.6 GPa (Γ∼1 N m−1) and 100 MPa (Γ=0.1 N m−1) for bubbles with radii R=5 nm and R=10 nm, respectively. The obtained adhesion energy for larger bubbles are in agreement with previous first-principles calculationsR∼1–10 nm) where they can experience a pressure of the order of GPa. Furthermore, nonlinear plate theory modifies equation where κ≅0.24 nN-nm is the bending stiffness of graphene. For hmax∼1 nm this gives P∼1.42 GPa (Γ=0.56 N m−1) and 89 MPa (Γ=0.035 N m−1) for R=5 nm and R=10 nm, respectively. It is seen that by including the second terms, pressure decreases with ∼10% and the adhesion energy with 50–80%. Notice that in κ varies slightly with temperaturehmax/R the contribution of the second terms in The second terms in In common MDs simulations2010fi). In where the first term is the ideal gas pressure and the second term is due to the interatomic potential force  and the vdW pressure (PvdW) induced inside a bubbleThe above drawbacks are overcome using the stress tensor-based methodσ, Vb, N, mu and NA are, respectively, the trapped atoms stress tensor in unit of pressure × volumewhere <.> represents a time average over several realizations and We emphasized that our method based on the virial stress tensor is more general than T=0 K. This is equivalent to previous approach based on elasticity theoryhmax and R. Subsequently, we increased T up to room temperature, and investigated the spatial structure of the trapped helium atoms. We found that the non-zero temperature results are different from the ground state results . At room temperature, thermal fluctuations occur in the trapped substance while the bottom graphene layer is supported by the substrate and therefore remained fixed. The bubble is found to fluctuate randomly and has in general a non-round shape. The inset of First, we simulate 792 helium atoms below the bumped graphene and found the optimized structure at z axis)Using lateral radial distribution function (RDF) along the direction perpendicular to the substrate  lattice structure of helium at 0 K which is consistent with the results reported by Hodgdon −3 and 0.65 GPa, respectively. Notice hmax and R given in Phyd=1.25 GPa. The latter difference is due to the non-round shape of the helium bubble. The obtained fluid helium density is comparable with experimental results (0.28 g cm−3) where a nonlinear increase of the fluid helium density was measured up to a pressure of 2 GPa at room temperatureUsing esults 0. g cm−3 wx- and y-directions and the RDF are given in Water is a polar liquid with a hydrogen bond network. We theoretically predicted previously the square-rhombic lattice structure for monolayer ice confined between graphene layersNext, we considered larger bubbles and compare them with typical bubbles measured in experiment. In z direction at two different temperatures 0 and 300 K. At 0 K, the trapped water molecules have a layered structure, that is, it shows three layers which are located at z=2.76, 5.66 and 8.36 Å. Surprisingly, confined water preserves its layered structure even at room temperature (representing the amorphous-solid-like phase for trapped water). The corresponding peaks are located at 2.82, 5.76 and 8.65 Å. At higher temperature, the inter-larger distance becomes larger. The density and the induced vdW pressure are estimated to be ρ=1.03 g cm−3 and PvdW=0.93 GPa. Again, due to the non-round shaped water bubble Phyd=1.4 GPa. The present study on the size effect shows the limitation of T=0 K. However, the top layers do not show any signature of the square ice structure and are less ordered than the bottom layer. In contrast to ref. A wide range of stable amorphous ice structures are possible due to the adaptability of perturbations in hydrogen bonded networks between the water molecules. All water layers exhibit a similar microscopic structure at room temperature, which can be seen from ρ=0.987 g cm−3 and PvdW=0.49 GPa, respectively. This is consistent with the fact that ethanol crystallizes for pressures around 1.5 GPa . Again, the size of the bubble is larger than our simulated sample, however the AFM images show a round shape with in-plane cylindrical symmetry in agreement with our MD results. More AFM images for hydrocarbon bubbles are presented in In In C16H34 known as hexadecane. We captured 160 molecules and optimized the structure. We found that they tend to be aligned with each other and form a bubble with a larger flat area. In Next, we performed additional simulations using large hydrocarbons, that is, R), maximum height (hmax), and aspect ratio (hmax/R) for the studied small nanosize bubbles (R<10 nm). We obtained hmax/R values between 0.17 and 0.20 which is larger than the experimentally reported value of 0.11 for large bubbles (R>10 nm) and it is in agreement with the experimental results on small size bubbleshmax/R ≅0.1) is not applicable for small size bubbles (R<5 nm).In We compared the density of trapped materials with those for bulk helium, water, and ethanol at room temperature at two different pressures and listed the results in σrr,σθθ) of the top graphene sheet for ethanol bubbles and found very good agreement with those predicted by membrane theoryσrr and σθθ as a function of radius obtained from our MD simulation for an ethanol bubble (radius∼4 nm) at 0 K. Stress components and radii are scaled to the maximum stress and bubble radius (R), respectively:Here, we study the radial and circumferential components of the stress tensor (σrr and σθθ stress components can be determined from fσ(x) which is a polynomial function of the radius (x<1) given byThe solid back curves in fσrr(x) and fσθθ(x) results in the values of 0.994 and 0.987, respectively  correspond to the cubic crystalline structure of NaCl which is not influenced by the vdW pressure in the nanocapillary. The inset of 31+and Cl− which is ∼1,000 kJ mol−1, two orders of magnitude larger than the hydrogen bond, that is, 10 kJ mol−1 in ice.In The aim of this section is to provide a closer look into the structure of the trapped materials inside the bubble and elaborate on their layered structure. It is important to note that different bubbles , regardless of their size and/or type, have perfectly optimized round shape at zero temperature. Even at higher temperature as the bubble gets smaller, it attains a more round-like shape. This is the reason why we obtained a non-circular shape for the large water bubble in agreement with AFM experiment , while iFinally, PvdW and ρ obtained from MD and elasticity theory. R<4 nm) the latter overestimates the pressure inside the bubble while for larger bubble radius the two approaches result in the same pressure. We believe that our simulated bubble would converge to the predicted pressure as obtained from elasticity theory in the limit of a large bubble. However, for a small bubble both membrane and nonlinear plate models predict unrealistic extremely high pressures. Note that barely noticeable difference between the two models is due to the small contribution of the bending rigidity (κ) to the elastic energy of graphene beyond the length scale of 4 Å —see The size of the bubble which is determined by the amount of trapped material has an impact on the physical properties of the bubble such as the density, the induced pressure, and the layered structure. To investigate this effect, we carried out different simulations in order to compare We presented an extensive atomistic MDs simulation study to probe the vdW pressure at atomically smooth graphene interfaces by intentional trapping different types of materials . The correlation between the shape of the graphene bubble and the physical properties of the trapped substances was pointed out. Temperature and concentration of trapped material have strong influence on the bubble formation. Our detailed simulations along with the experimental observations provides insightful information about the formation of bubbles (including flat nanobubbles) and the effect of vdW pressure on the structural and conformational changes of the trapped substance.We found that the bump shape and height depend on the thickness and elastic properties as well as on the specific trapped molecules. Experimental investigations on the geometrical and physical properties of graphene bubbles filled with different substances have motivated us to perform extensive atomistic simulations to determine the microscopic structure of the trapped substances and the corresponding deformation of the graphene flake. We tested five different substances namely, helium, water, ethanol, hexadecane and NaCl inside the graphene nanobubble with an effective radius smaller than 10 nm using appropriate interatomic potentials. Significant differences in the microscopic details of the bubbles were found for different encapsulated substances. Trapped helium behaves like a gel with the highest degree of fluidity subjected to a pressure of 0.65 GPa, while NaCl is very rigid and resists against pressures of order GPa and forms a flat like bubble in contrast to the bubbles found for helium, water and ethanol. On the other hand, helium exhibits a layered structure only at low temperature while water and ethanol preserve their layered structure even at room temperature. The deformed graphene over NaCl follows the shape of the NaCl crystal, except if it in solution. An amorphous and layered structure of water was found even when subjected to 0.93 GPa pressure at room temperature. The water nanobubbles do not have a perfect round shape. Ethanol behaves like a liquid at room temperature, while it forms an amorphous solid at 0 K. The deformation profile for bubbles filled by hydrocarbons (ethanol) are more relevant to our AFM images and the recently reported graphene nanobubblesIn 33Moreover, we have shown that even by increasing the adhesion energy between two graphene sheets, which is equivalent to increasing the vdW pressure, a more ordered structure for water is obtained, while the square ice structure and its stacking structure cannot be seen see . We founεHe=0.02166, kcal mol−1, σHe=2.64 Å) as well as the helium–carbon  interactionsWe used MD simulations using reactive force field ReaxFFet al.39To simulate trapped ethanol, we used the hybrid optimized potential for liquid simulations (OPLS) potentialεNa−C=0.006505, kcal mol−1, σNa−C=2.78 Å, εCl−C=0.001153, kcal mol−1, σCl−C=4.115 Å)For trapped NaCl nanocrystal simulations, we used hybrid potentials including AIREBOOur simulation setup comprises three different parts: (i) the square shape substrate which is a rigid graphene sheet with 120,000 carbon atoms, (ii) a circular flake of graphene with 72,000 atoms with a typical radius of 17 nm and (iii) the trapped molecules, see 2/Si substrate. A well-controlled micro-manipulation setup was used to transfer the top layer for successful encapsulation of the solvent. After placing the single-layer graphene on top of 2 μl solvent, most of it was spontaneously squeezed out by leaving only a very small amount in between the top and bottom graphene layers. Overnight drying of the prepared samples at room-temperature led to the gradual evaporation of the solvent which allows the top graphene layer to completely collapse onto the bottom graphene flake with a tiny amount of solvent captured in between. These samples were placed in vacuum (∼1 mbar) for few hours before removing the PMMA layer using acetone wash. As prepared sandwich samples, containing the water/ethanol filled graphene nanobubbles, were used for atomic force microscope imaging using Bruker Dimension Fastscan AFM operating in peak force tapping mode. In-plane radius of the bubble in this study refers to the base radius measured using AFM profile. Height profile is taken across the bubbles by allowing the measuring line to pass through the centre of the bubble.Graphene nanobubbles filled with water/ethanol molecules were fabricated by wet transfer technique using single and few layer graphene flakes (50 μm × 50 μm or above) prepared on the oxidized silicon substrate via mechanical exfoliation, as described previouslyThe data that support the findings of this study are available from the corresponding author on request.How to cite this article: Ghorbanfekr-Kalashami, H. et al. Dependence of the shape of graphene nanobubbles on trapped substance. Nat. Commun.8, 15844 doi: 10.1038/ncomms15844 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Even with recent advances in therapy regimen, multiple myeloma patients commonly develop drug resistance and relapse. The relevance of targeting the PD-1/PD-L1 axis has been demonstrated in pre-clinical models. Monotherapy with PD-1 inhibitors produced disappointing results, but combinations with other drugs used in the treatment of multiple myeloma seemed promising, and clinical trials are ongoing. However, there have recently been concerns about the safety of PD-1 and PD-L1 inhibitors combined with immunomodulators in the treatment of multiple myeloma, and several trials have been suspended. There is therefore a need for alternative combinations of drugs or different approaches to target this pathway. Protein expression of PD-L1 on cancer cells, including in multiple myeloma, has been associated with intrinsic aggressive features independent of immune evasion mechanisms, thereby providing a rationale for the adoption of new strategies directly targeting PD-L1 protein expression. Drugs modulating the transcriptional and post-transcriptional regulation of PD-L1 could represent new therapeutic strategies for the treatment of multiple myeloma, help potentiate the action of other drugs or be combined to PD-1/PD-L1 inhibitors in order to avoid the potentially problematic combination with immunomodulators. This review will focus on the pathophysiology of PD-L1 expression in multiple myeloma and drugs that have been shown to modulate this expression. Multiple myeloma (MM) is the second most common hematological malignancy . Even wiProgrammed death-ligand 1 (PD-L1), also known as B7-H1 and CD274, is a cell-surface glycoprotein that links to receptor programmed cell death-1 (PD-1) on T lymphocytes. It is normally involved in peripheral tolerance –7. It isThe relevance of targeting the PD-1/PD-L1 axis in MM has been demonstrated in pre-clinical models, and many clinical trials are ongoing. There are two major PD-1 inhibitors being studied in MM: nivolumab and pembrolizumab. There is one major PD-L1 inhibitor currently studied in MM: durvalumab. Table There have recently been concerns about the safety of PD-1 inhibitors in combination treatments with immunomodulators for MM. Based on these concerns, several clinical trials involving PD-1 and PD-L1 inhibitors combined with pomalidomide and lenalidomide have been suspended or put on clinical hold pending further results. Trials evaluating combination of PD-1/PD-L1 inhibitors with other classes of drugs are still ongoing. There is therefore a justification for exploring alternative combinations of drugs or different approaches to target this pathway. The rationale for the adoption of new strategies targeting the expression of PD-L1 on MM cells will be reviewed, and new therapeutic avenues will be discussed.A number of studies have demonstrated that PD-L1 expression is present in plasma cells from patients with MM, but not from healthy donors –13, and There is a large body of literature addressing the mechanisms that regulate PD-L1 expression in cancer cells , 18–21. Other regulatory pathways of PD-L1 have been shown to be involved in MM. Production of IL-6 by stromal cells is of relevance in MM, as evidenced by the induction of PD-L1 by IL-6 on human myeloma cell lines, which can be downregulated by inhibiting STAT3, MEK1/2, or JAK2 . PD-L1-iStudies in cell lines and animal models have provided sufficient evidence for the potential therapeutic effect of PD-1/PD-L1 axis blockade in MM , 13, 16.PD-L1 can induce T-cell apoptosis in vitro and in vivo via binding to PD-1 . The binTamura et al. showed that RPMI8226 and KMS-34 MM cells expressing surface PD-L1 had a proliferative advantage over cells from the same line that did not express PD-L1, as demonstrated by higher fraction of cells in the G2/M phase, higher levels of BrdU incorporation, percentage of Ki67 positivity, and more rapid proliferation in cell cultures . ConversThe effect of PD-L1 overexpression on cell proliferation was also identified in other types of cancer cells, such as colorectal carcinoma cell line, gastric cancer cell lines, pancreatic cell lines, as well as an ovarian carcinoma cell line –53. OverIn RPMI8226 cells, PD-L1+ cells had markedly higher expression levels of Bcl-2 and FasL expression compared to PD-L1- cells. The levels of intracellular Bcl-2 and cell-surface FasL proteins were also higher in PD-L1+ cells . ConversUsing standard Matrigel-coated or uncoated transwell chamber assays, Shi et al. showed that knockdown of PD-L1 in colorectal cancer cell line, HCT116, was associated with reduced migration and invasion ability, with a reduced invasive index . SimilarDexamethasone and melphalan induced marked apoptosis in PD-L1- RMPI8226 cells, but not in PD-L1+ cells. Apoptosis was also observed in KMS-28PE cells that did not express PD-L1, but not in PD-L1-transduced cells . ConversHigher expression of PD-L1 was associated with a higher percentage of infiltration of the bone marrow in MM patients. PD-L1 expression levels were also often upregulated in patients that relapsed or became refractory to therapy . In a prMultiple studies have demonstrated the prognostic significance of PD-L1 expression by IHC in solid tumors –63 and in = 5), progressed (n = 5), and stable (n = 4), the authors showed that early progression was associated with lower basal NK cells numbers and a lower PD-1 expression in effector memory CD8 cells [These factors also complicate the assessment of the value of PD-L1 IHC for predicting response to immune checkpoint blockade. While the expression of PD-L1 by IHC is generally associated with a higher proportion of objective response,  patientsD8 cells .A clinical study combining Pembrolizumab, Pomalidomide, and low-dose dexamethasone in relapsed/refractory MM examined PD-L1 expression in bone marrow biopsy of 29 patients by IHC, using a double staining with MUM1 to facilitate identification of plasma cells. There was no correlation with outcome; however, this is a small sample with a median follow-up of 15.6 months. To our knowledge, this was the first study to evaluate PD-L1 in bone marrow using IHC . The cut+ T cells [PD-L1 impacts prognosis not only through inhibition of immune response, but also by generating intrinsic aggressive characteristics in MM cells . As desc T cells . Both le T cells , or lymp T cells , have alA recent review by Jelinek et al. highlighted the various clinical trials that are ongoing for PD-1/PD-L1 inhibitors in hematological malignancies including MM . PublishThere were however recent developments that might affect the future of trials involving PD-1 and PD-L1 inhibitors. In June 2017, Merck and the Food and Drug Administration (FDA) suspended enrolment for randomized trials of pembrolizumab with pomalidomide and lenalidomide in relapsed and newly diagnosed patients, based on an imbalance of deaths in the pembrolizumab arms. Two trials (KEYNOTE-183/NCT02576977 and KEYNOTE-185/NCT02579863) were put on full clinical hold due to risks that outweighed any potential benefit for patients , 85. IntTrials combining immunomodulators with anti-PD-L1 inhibitors durvalumab (NCT02685826) and atezolizumab (NCT02431208), as well as PD-1 inhibitor nivolumab (NCT02903381), were then suspended based on the safety concerns raised from pembrolizumab trials . SeveralConsidering the concerns regarding the combination of PD-1/PD-L1 inhibitors with immunomodulators, new strategies are needed to target this pathway. Several molecules have been shown to modulate PD-L1 expression on tumor cells, which can be exploited as monotherapies, combined to other drugs to help potentiate their action or combined to PD-1/PD-L1 inhibitors. The following sections will focus on selected molecules that have been investigated in MM and have shown promising results Table .Table 3DOncolytic virus therapy is a therapeutic strategy that has gained interest in the treatment of MM . ReolysiHistone deacetylase (HDAC) inhibitors are a new class of drugs that has shown promising results in the treatment of MM. There are 70 clinical trials involving HDAC inhibitors in MM listed on clinicaltrials.gov as of 2 January 2018 . InteresA study also showed that pan HDAC inhibitors sensitize MM cells to Reolysin in vitro and in vivo by increasing the expression of reovirus receptor JAM-1. This combination increased reovirus replication in MM cells and was associated with significantly decreased bone marrow infiltration in a mice model . Since sBCMA is a selective antigen found on plasma cells and plasmacytoid dendritic cells, making it an interesting target for the treatment of MM . It has Conversely, MEK1/2 inhibitor U0126 was able to block APRIL-induced PD-L1 expression in MM cell lines . U0126 cBecause of its relatively selective expression in plasma cells, BCMA has gained interest as a target for chimeric antigen receptor (CAR) T cell therapy. CAR T cells are synthetically engineered T cells that express artificial CARs that target a tumor cell surface molecule. They can therefore recognize tumor cells even if they do not express human leucocyte antigen molecules . There aPancreatic tumor cells treated with mimics of miR-93-5p and miR-106b-5p showed reduced expression of PD-L1. This effect could be reproduced by treatment with bromodomain and extraterminal (BET) inhibitor OTX015 . This waA STAT3 inhibitor, WP1066, could abrogate PD-L1 expression in lymphoma cell lines, leading to decreased cell growth and increased cell apoptosis . There iIbrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), caused selective and durable downregulation of PD-L1 on chronic lymphocytic leukemia cells within 3 months of therapy, which was mediated through the inhibition of constitutively active STAT3 on these cells . The comhigh drug-resistant cells to chemotherapy, thereby suggesting a role for miRNA replacement therapy [MiRNAs represent another class of molecules that have gathered interest as a target for cancer therapy. A comprehensive review of miRNAs involved in PD-L1 regulation is outside the scope of this paper; however, there have been two recent reviews on the topic by Wang et al. and Smolle et al. , 113. So therapy .To our knowledge, miRNAs involved in regulation of PD-L1 in MM have yet to be described; however, considering the overlap with some miRNAs known to be involved in MM, such as the miR-15 family, the miR17~ 92 cluster, miR-21, miR-34a, or miR-106b , 119, itNotably, treatment of acute myeloid leukemia cells with MUC1-C inhibitor peptide (GO-203) caused an increase in miR-34a and miR-200c, which lead to a decreased expression of PD-L1 and decreased leukemia involvement in an animal model . G0-203 Interesting results have been obtained in clinical trials combining PD-1 inhibitors with immunomodulators in MM; however, concerns regarding the safety of these combinations have been raised and several clinical trials have been suspended or put on hold. There has been increasing interest in new therapeutic strategies directly targeting the expression of PD-L1 on cancer cell surface, which could be a relevant approach in MM. Further studies aiming to target the molecules involved in the regulation of PD-L1 in MM will allow the design of new drugs or the application of available drugs that can affect the expression of PD-L1. While PD-L1 expression is relatively restricted in normal tissue, targeting downstream molecules involved in its regulation could potentially be associated with more widespread adverse effects. This will need to be addressed in ongoing and future clinical trials. However, promising results have been obtained in clinical trials with CAR T cells targeting BCMA, HDAC inhibitors, and oncolytic reovirus, supporting the relevance of these alternative strategies in the treatment of MM. As research on the PD-1/PD-L1 axis continues, a variety of molecules are found to have a role in the regulation of PD-L1 expression, such as Sigma1 inhibitor IPAG , serine


Aloe vera and Azadirachta indica (neem) during pre-starter age (0-2 weeks) on gut health and histomorphometry in Vanaraja chicks.This study was conducted with an aim of studying the efficacy of water supplements of Aloe juice per chick per day, T3: 3 ml neem extract per chick per day, T4: 1.5 ml Aloe and 1.5 ml neem per chick per day. Gut culture was done for Escherichia coli and Lactobacillus sps. and gut histomorphometry in 24 gut samples at 14 days of age.A total of 192 day old Vanaraja chicks were randomly assigned to one of four herbal water treatments throughout the experimental pre-starter stage (0-2 weeks) in a completely randomized design. Each treatment was given four replicates consisting of 12 chicks per replicate. Water treatments comprised T1: Control with regular antibiotic supplement, T2: 3 ml A. vera and neem in water significantly (p<0.05) reduced and increased the number of gut E. coli and Lactobacillus sps. Colonies, respectively, as compared to that of control groups; Villi was significantly (p<0.05) taller and broader on 14 days of age across the jejunum of chicks fed with neem supplementation as compared to that of control chicks. Significantly lower crypt depth (p<0.05) was observed in the duodenum of Aloe supplementation. Villus height: Crypt depth ratio of duodenum and jejunum was significantly (p<0.05) increased neem and Aloe supplementation in chicks as compared to their combination and control.This study revealed that supplementation of Immediate post hatch supplementation of Aloe juice and neem extract in chicks improved the development and health of their gut. Early chick nutrition, commonly termed as pre-starter feeding helps in utilization of yolk sac for optimal immunity and gut development and to enhance its function. Early chick feeding has a great potential to trigger the gut and its morphological development for improved performance and better feed conversion ratio ,2. HenceAloe vera has a rich source for many chemical compounds and plays various roles in animal system. Similarly, Azadirachta indica (neem) leaves have vast properties such as as immune modulatory, anti-inflammatory, antihyperglycemic, antimalarial, antifungal, antibacterial, antiviral, antioxidant, antimutagenic, and anticarcinogenic [Aloe and neem juice on gut histomorphology is limited.Medicinal plants have an indispensable source of medicine for poultry production systems since ancient times. A significant number of farmers are still judiciously using herbal remedies in the management of rural poultry in spite of modern veterinary treatment. As per the estimation of WHO, even today 80% of people still rely on medicinal plants  for theiinogenic -8. Neem inogenic . HoweverA. vera and A. indica during pre-starter age (0-2 weeks) on gut histomorphometry of Vanaraja chicks.The morphology of intestinal villi and crypts has been associated in chickens with intestinal function and growth. Adverse changes in the content of the digesta, such as high population of pathogenic bacteria, parasites or substances, could lead to changes in the surface of intestinal mucosa, because of their close proximity. Hence, the aim of this work is to study the efficacy of water supplementation of The experiment was conducted after the permission of Institutional Animal Ethics Committee of ICAR-Central Island Agricultural Research Institute, Port Blair.A. vera and A. indica (neem) were freshly collected for the study. A. vera juice was prepared by grinding the pulp without water, filtering and mixing with water at 1:1 ratio; 200 g of neem leaves in 1 L of water was kept in shaking water bath for overnight for extraction and was filtered in the morning. The pre-starter feed was prepared with 20% crude protein and 2800 kcal ME/kg as per BIS (2007) recommendation and fed ad libitum to the experimental chicks. A total of 192-day-old Vanaraja chicks were randomly assigned to one of four herbal water treatments throughout the experimental pre-starter stage (0-2 weeks) in a completely randomized design. Each treatment was given four replicates consisting of 12 chicks per replicate. Water treatments comprised T1: Control with regular antibiotic supplement, T2: 3 ml Aloe juice per chick per day, T3: 3 ml neem extract per chick per day, T4: 1.5 ml Aloe juice and 1.5 ml neem extract per chick per day.th day of age. The gut contents were cultured for Escherichia coli and Lactobacillus sps. in nutrient broth and MacConkey Rogosa (MRS) broth, respectively, and identified using specific respective medias of eosine methylene blue and MRS agar. The microbial counts were determined as colony forming units per gram of samples (n=6 per treatment). The histomorphological study of tissue samples (n=6 per treatment and 6 fields per sample) was carried out according to the method described by Bancroft and Marilyn [Tissue samples from duodenum, jejunum and ileum and internal contents of intestine and ceca were collected from six Vanaraja birds by slaughtering, from each treatment by humane method at 14 Marilyn . The dat Marilyn . The sig Marilyn .A. vera and neem in water significantly (p<0.05) reduced and increased the number of gut E. coli and Lactobacillus sps. colonies, respectively, as compared to the control  broader width was recorded in jejunum at 14Aloe and Aloe plus neem supplementation as compared to control. Significantly lower crypt depth (p<0.05) was observed in the duodenum when the Aloe was supplemented as compared to neem and Aloe plus neem supplementation. Water supplementation of Aloe, neem and their combination did not result in significant differences in the crypt depth of jejunum segment. Villus height: Crypt depth ratio of duodenum and jejunum was significantly (p<0.05) increased at 14 days of neem and Aloe supplementation in chicks as compared to their combination and control. Small lumen and epithelium (Plate-1) were observed in the intestinal glands attached to the villi of control chicks. The inter-glandular corrion connects both the lymphatic and capillary network and was loaded with many infiltrate cells. The capillary network showed evidence of hyperplasia and hypertrophy, whereas the chicks from Aloe and neem supplementation had villi with intestinal glands of the duodenum having a large lumen and were surrounded by thin interglandular spaces, with the interglandular villi containing collagen fibers, fibroblasts and leukocytes infiltrate. The presence of the capillary ectasia in the main villi and interglandular villi in the present images of duodenum suggest that the angiogenesis process has been stimulated. The capillary network underwent both hyperplasia and hypertrophy and seen to be powerfully stimulated by the lymphoid infiltrate.Significantly (p<0.05) lower crypt depth was seen with neem and A. vera [Lactobacillus spp. and reduced E. coli count through supplementing the feed with A. vera gel and Poonam et al. [Lactobacillus sps. in that intestine. The final fermentation product of Lactobacillus sps. is lactic acid that might has made the gut environment unfavorable for pathogens and modified harmful microbial population.The microbial population in the gastrointestinal tract of poultry plays an important role in normal digestive processes and health maintenance . This miA. vera . The resA. vera  who alsom et al.  as well Aloe supplementation significantly (p<0.05) improved the height of villi in duodenum, jejunum and ileum  significantly reduced by Aloe and neem supplementation in duodenum and ileum except jejunum. Significantly higher villi and lower crypt depth increased their ratio in the duodenum and jejunum of chicks fed with Aloe and neem extract. Water supplementation of Aloe, neem and its combination did not bring out any significant differences in the villi height and depth ratio of illeum segment. The results of present study are in agreement with the report of Homan et al. [et al. [Aloe and neem supplementation has made the gut environment free of microbial toxins [Lactobacillus sps. in that intestine are responsible for favorable change in intestinal morphology and might have stimulated the proliferation of epithelial cells of the bowel [Aloe and neem supplementation indicated for slow tissue turnover preventing the pathogens from tissue destruction in the gut [It was observed that neem and n et al. , Kadhim  [et al.  that lon [et al. . Accordil toxins . Short-che bowel . In addi the gut .Aloe and neem in the water of Vanaraja chicks enhanced the gut health and gut development. The A. vera and A. indica might be promising replacers to antibiotic growth promoters for organic poultry production.Based on this comprehensive study, it is concluded that immediate post hatch supplementation of extracts of TS contributed in designing of experiment extraction and feeding experiment in poultry. SA did the feeding experiment, monitoring of the experimental birds. JS did the microbial analysis of gut flora. MT did the histopathological analsyis of gut tissues. AK did the analysis and overall monitoring of the experiment and preparation of manuscript. All authors read and approved the final manuscript.


The impact of serum levels of soluble programmed cell death ligand 1 (sPD-L1) on prognosis in patients with Epstein-Barr virus-associated malignancies has never been investigated. We prospectively measured pre- and post-treatment serum sPD-L1 levels and evaluated their prognostic value in 97 patients with newly diagnosed, early stage extranodal NK/T-cell lymphoma (ENKTCL) treated with asparaginase-based chemotherapy followed by radiotherapy. For predicting survival outcomes, serum sPD-L1 levels of 3.23 ng/mL and 1.12 ng/mL were respectively identified for pre- and post-treatment cut-off levels. Patients with high pretreatment (>3.23 ng/mL) had shorter progression-free survival (PFS) and overall survival (OS). In a multivariate survival analysis, post-treatment sPD-L1 >1.12 ng/mL, treatment response (complete vs. non-complete response), and stage II disease were independent prognostic factors for shorter PFS and OS. In patients with a complete response, post-treatment sPD-L1 >1.12 ng/mL was associated with shorter PFS and OS. In patients with high pretreatment sPD-L1 levels (>3.23 ng/mL), low post-treatment sPD-L1 level (≤1.12 ng/mL) correlated with longer PFS and OS. Our data suggest the post-treatment sPD-L1 level is a potent biomarker for predicting early relapse and poor prognosis in early stage ENKTCL patients treated with asparaginase, and may be a useful marker of minimal residual disease. Extranodal NK/T-cell lymphoma (ENKTCL) is an aggressive non-Hodgkin's lymphoma  and is cet al. recently reported that the soluble PD-L1 (sPD-L1) concentration in blood could predict overall survival and treatment response in diffuse large B cell lymphoma (DLBCL) . According to this cutoff value, 63 patients (64.9%) were assigned to the low-sPD-L1 group (≤3.23 ng/L), and 34 patients (35.1%) were assigned to the high-sPD-L1 group (>3.23 ng/L). The baseline clinical characteristics of the patients in the low- and high-sPD-L1 groups are compared in Table (P = 0.639).The median serum sPD-L1 concentration for all patients was 2.76 ng/mL , and the mean was 2.79 ng/mL. Serum sPD-L1 was measured in twenty healthy volunteers, and the median of 0.68 ng/mL (range 0.057–1.30 ng/mL) was lower than that of ENKTL patients (P = 0.008), as shown in Table P<0.001, Figure All patients received asparaginase-based chemotherapy followed by radiotherapy. The GELOX regimen was used in 71 patients 73.2%), and the remaining 26 patients (26.8%) received CHOP-L chemotherapy. After the initial treatment, 76 (78.4%) of the 97 treated patients achieved CR, 18 patients (8.8%) had a partial response (PR), and 3 patients had stable disease (SD). The CR rate at the end of treatment was higher in the low-sPD-L1 group than in the high-sPD-L1 group , the 3-year PFS and OS rates for all 97 patients were 65.0% (95% CI 55.2–74.8%) and 78.8% (95% CI 70.0–87.6%), respectively. Patients in the high-sPD-L1 group had shorter PFS . In those achieving CR, patients with post-treatment sPD-L1 levels>1.12 ng/mL had shorter PFS and OS Figure . MeanwhiP<0.0001, P<0.001).We investigated the presence and distribution of PD-L1 in ENKTCL tissue using immunohistochemistry. At least 5% of tumor cells were PD-L1-positive in 76 out of the 97 patients . The perIn this study of ENKTCL patients receiving an asparaginase-based chemotherapy regimen, we found that high pre-treatment sPD-L1 levels were associated with poor treatment response and post-treatment sPD-L1 level was an independent prognostic factor for both PFS and OS. Specifically, post-treatment sPD-L1 levels identified patients with a CR after treatment who had a high risk of relapse and poor prognosis. Therefore, we propose that post-treatment sPD-L1 levels are a useful biomarker for monitoring minimal residual disease (MRD) in ENKTCL patients.Previous studies have demonstrated the role of PD-L1 in various solid tumors, including lung cancer, breast cancer, and nasopharyngeal carcinoma –19. A clin vitro studies, we found that ENKTCL cell lines with high PD-L1 expression were less susceptible to pegaspargase and gemcitabine than those with low PD-L1 expression  untreated, histologically confirmed ENKTCL patients; (2) primary tumor site located in the upper aerodigestive tract; (3) Ann Arbor stage was IE or IIE; (4) complete treatment and follow-up data; (5) available serum collected at the time of diagnosis and after treatment. The exclusion criteria were: (1) previous or concomitant malignancies; (2) previous anti-cancer treatments; (3) any coexisting medical problems that could cause poor compliance with standard antitumor therapy protocols.Clinical data collected from patients included the following information: patient demographics, Eastern Cooperative Oncology Group performance status (ECOG PS), physical examination, serum LDH, B symptoms, examination of bone marrow smear, bone marrow trephine biopsy, nasal and oral cavity endoscopic examination, head and neck magnetic resonance imaging (MRI), and positron emission tomography-computed tomography (PET-CT) of the whole body. The standard Ann Arbor staging system was used to determine stage for all patients, as calculated by the International Prognostic Index  and NK/T cell lymphoma Prognostic Index  .All patients were treated with upfront asparaginase-based chemotherapy, e.g. GELOX   and CHOPBlood samples were obtained from the 97 patients at diagnosis and 1 month after the completion of treatment and from 20 healthy volunteers from the Guang Zhou City Blood Bank matched for sex and age with all enrolled patients. Serum was collected from the blood samples by centrifuging at 4000 × g and stored as 500 μL aliquots at −80°C until assays were conducted. Sandwich enzyme-linked immunosorbent assay (ELISA) kits  were used to determine serum soluble PD-L1 level according to the manufacturer's protocol. The minimum detectable level of sPD-L1 was 0.057 ng/mL. Briefly, samples and standards were added to a microplate precoated with a PD-L1-specific monoclonal antibody. After enzyme reagent and any unbound antibody was removed by washing, a substrate solution was added to the wells, Stop Solution was used to terminate color development, and the absorbance value was read at 450 nm using a spectrophotometer . The sPD-L1 concentrations were calculated using a standard curve, which was constructed using the standards.Representative formalin-fixed, paraffin-embedded tissues obtained from surgical resections or biopsies were used for IHC. Four-micrometer-thick sections ofparaffin-embedded tissues were cut, placed on slides, deparaffinized in xylene, and hydrated in a graded alcohol series. Immunohistochemical staining of PD-L1 was performed by incubating with a 1:50 dilution of a PD-L1 antibody . IHC was performed using a modified avidin-biotin peroxidase complex amplification and detection system. PD-L1 expression percentages were quantified by determining the number of cells with membrane staining among the total number of tumor cells in the field examined under high magnification (200x).P value was <0.05, variables were considered significant in univariate analysis and were entered into multivariate analysis. Multivariate analysis using the Cox regression model was used to evaluate the impact of selected variables on prognosis.Serum sPD-L1 levels are expressed as median , while categorical data are presented as n (%). Correlations between pretreatment serum sPD-L1 levels and clinical characteristics were analyzed using Mann–Whitney U tests or Chi-square tests. The cutoff levels for pretreatment sPD-L1 and post-treatment sPD-L1 for survival prediction were determined using the receiver operating characteristics (ROC) curve analysis. The Kaplan-Meier method and log-rank test were used for survival analysis. If the two-sided


In this study, we report a novel strategy to prepare graphene nanopapers from direct vacuum filtration. Instead of the conventional method, i.e., thermal annealing nanopapers at extremely high temperatures prepared from graphene oxide (GO) or partially reduced GO, we fabricate our graphene nanopapers directly from suspensions of fully reduced graphene oxide (RGO), obtained after RGO and thermal annealing at 1700 °C in vacuum. By using this approach, we studied the effect of thermal annealing on the physical properties of the macroscopic graphene-based papers. Indeed, we demonstrated that the enhancement of the thermal and electrical properties of graphene nanopapers prepared from annealed RGO is strongly influenced by the absence of oxygen functionalities and the morphology of the nanoflakes. Hence, our methodology can be considered as a valid alternative to the classical approach. The unique physical properties of this carbon allotrope [Two-dimensional 2D) materials, i.e., one-atom thick layers of van der Waals materials, have gained worldwide attention because of their outstanding properties that arise from their structure and dimensionality ,2. GraphD materiallotrope ,4,5,6,7,llotrope ,9,10, hallotrope ,13,14,15sp2 hybridization of graphene layers, deteriorating the thermal and electrical conductivities of the nanoflakes and, in turn, reducing the performance of the as-prepared GO papers.Recent advances in the development of flexible graphene paper-like architectures with superior thermal and electrical properties have focused their attention on different assembly strategies ,14,15,16N-methylpyrrolidone (NMP), the addition of surfactants to the aqueous medium or the formation of graphene colloids. Despite the interactions between oxygen-bearing groups are limited, the vacuum filtration-induced directional-flow allows the assembly of RGO sheets into well-ordered macroscopic structures with remarkable mechanical and electrical properties [In order to restore the π-conjugated system of these materials, two different approaches have been reported. The first strategy is based on the reduction of the as-prepared GO films by chemical methods (using hydrazine hydrate or hydriodic acid), thermal processes or a combination of both. As a result of these processes, the conductivities are partially recovered due to the gradual removal of the oxygen species and water moisture between the nanosheets, packing them more tightly ,19,25,26operties ,29,31.We have recently reported that the thermal properties of RGO flakes after thermal annealing at 1700 °C are increased due to the reduction of oxygen-functional groups and the ordering of the graphene structure . This ha−1 and the solutions were sonicated in pulsed mode (15 s on and 15 s off) for 15 min with power set at 30% of the full output power (750 W), to avoid significant temperature increase during the treatment, by using an ultrasonication probe  with a 13 mm diameter Ti-alloy tip. The RGO and RGO_1700 suspensions were subjected to vacuum filtration using a Nylon Supported membrane . After filtration, the as-obtained papers were peeled off from the membranes and dried at 65 °C under vacuum for 2 h to completely remove the solvent. Finally, RGO and RGO_1700 nanopapers were mechanically pressed in a laboratory hydraulic press  under a uniaxial compressive load of 5 kN for 10 min at 25 °C.RGO and RGO_1700 flakes were prepared accordingly with the previously reported method , consistα) was measured using the xenon light flash technique (LFT) . The method is compliant with the international standard methods ASTM E-1461, ASTM E-2585, DIN 30905 and DIN EN-821. The samples were cut in disks of diameter 23 mm and the measurements were carried out in a special in-plane sample holder, in which the sample is heated in the central region and the temperature rise is measured on the outer ring of the sample. Measurements were carried out twice using different nanopapers for both RGO and RGO_1700. Each of the samples was measured five times: deviation between diffusivity values was typically found within ±5%. The in-plane thermal conductivity of the film, k, was then calculated from the equation k = ρ × α × Cp, where ρ is the density of the graphene nanopaper and Cp is the specific heat capacity of graphite (Cp = 0.71 J (g·K) −1) [ρ) of the samples was calculated according to the formula ρ = m/V, where m is the mass of the nanopaper, weighed using a microbalance (sensitivity: <0.1 µg) and V is calculated from a well-defined disk film using the average thicknesses measured as described in [The morphology of the graphene papers was characterized by a high resolution Field Emission Scanning Electron Microscope . The thermal diffusivity (g·K) −1) . The nankn, is calculated from Equation (1), where k is the thermal conductivity of the nanopaper, kair is the thermal conductivity of air and φ is the porosity of the nanopapers, i.e., the volume fraction of air, calculated as φ = 1 − (ρ/ρg), where ρg = 2.2 g·cm−3 is the density of graphite [In order to compare the thermal conductivity obtained on nanopapers with different density, owing to different porosity, the conductivity of dense nanoflakes networks were obtained by applying the well-known Maxwell’s effective medium approach . Therefographite .(1)k=knR is the measured resistance, d the distance between the voltage contacts and S = t × w where t and w are the thickness and width of the sample, respectively. Conductivity calculation include φ to compensate for different densities of the nanopapers, accordingly with the method applied for the thermal conductivity calculation. Uncertainty was evaluated by the propagation of experimental errors on the several measurements used in the calculation and sample inhomogeneity. The most important contributions to the uncertainty of the calculated conductivity values are represented by the deviations in the distance between the voltage electrodes Δd, in the range of ±0.3 mm, due to the finite width of the silver paste spots and the thickness of the nanopapers, Δt, in the range of ±2 µm. A conservative error band was thus estimated in the range of ±15–20%, depending on the specimen quality.The electrical conductivity was measured at room temperature by the standard four-point probe method. In order to have a regular geometry, the nanopapers were cut into rectangular stripes with nominal width of 4 mm. The electrical contacts were then fabricated by means of a small drop of conductive silver paste and connected to an Agilent 34420A Nanovoltmeter . The conductivity was calculated by the formula RGO and RGO_1700 were synthesized and characterized as described in our previous work . RGO and−3. The RGO_1700 nanopaper  and 100 °C (35 ± 2 mm2·s−1), which is expected owing to phonon Umklapp-scattering [2·s−1 for the RGO_1700 nanopaper was obtained at 25 °C, clearly evidencing a better performance of the nanopaper obtained with annealed RGO nanoflakes. However, due to the significant differences in density and thickness between nanopapers prepared with pristine and annealed nanoflakes, such figures represent the performance in heat spreading on the different nanopapers  and not the intrinsic property of the dense network made of RGO/RGO_1700 nanoflakes. While the in-plane thermal conductivity of the nanopaper can simply be calculated from in-plane diffusivity measurements multiplied by its density and heat capacity, a comparison between the intrinsic thermal conductivity of the dense networks made of RGO or RGO_1700 should be made by decoupling from the nanopaper density. Conductivity values of the nanoflake networks were therefore obtained by applying the Maxwell’s effective medium approach, as detailed in the experimental section.In-plane thermal diffusivity for both RGO and RGO_1700 nanopapers was measured by xenon flash method. The RGO nanopaper showed a diffusivity value of 43 ± 2 mmattering . A diffu−1·K−1 for the RGO nanopaper and 390 W m−1·K−1 for the RGO_1700 one at 25 °C, thus evidencing a dramatic increase of thermal transfer performance of the carbon nanoflakes network upon high-temperature annealing of the RGO nanoflakes. The enhancement of the thermal dissipation of the individual RGO flakes after thermal annealing, as a consequence of the removal of oxygen-functional groups with a simultaneous restoration of the sp2 hybridization of the C atoms, has already been proved by Scanning Thermal Microscopy (SThM) [The in plane thermal conductivity values of the dense nanoflakes networks were calculated according to this model giving, within ±10% error, 95 W·my (SThM) , even thy (SThM) . Here weBeside the effect of annealing, it is worth noting that the thermal conductivity of the nanopaper prepared in this work by filtration of RGO is higher to that reported by Renteria et al. for their dense rGO nanopaper prepared by deposition of GO , thermal3 S·m−1 for the RGO flakes, to (2.1 ± 0.4) × 104 S·m−1 for the annealed ones. These results are in accordance with the improvements observed in the thermal properties. It is known that defects and incomplete reduction hinder the electrical performance of RGO, in terms of both reduced electrical conductivity and charge carrier mobility and the thermal treatment favours the increase of sp2 domains and thus the graphitization of the nanoflakes [3 and 5.0 × 103 S·m−1, respectively [We further studied the electrical conductivity of the RGO and RGO_1700 nanopapers using the four-point probe method. Electrical conductivities measured were normalized on RGO flakes volume fraction, accordingly with the method applied in thermal conductivity. The electrical conductivity increases from (5.8 ± 0.7) × 10noflakes ,42. In tectively ,43, respsp2 carbon structure but also generates the formation of higher density structures. The combination of the thermal and electrical properties accompanied by the low density of these macroscopic graphene-based materials makes these assembled architectures potential candidates in thermal management and electronic applications.In summary, our procedure to prepare graphene-based nanopapers by flow-directed filtration-induced technique using mild conditions and without post-treatments is effective to produce highly porous and flexible macroscopic all-graphene based structures. The structure of the resultant graphene nanopapers is strongly influenced by the defectiveness of the starting RGO flakes. The thermal and electrical properties were improved in nanopapers prepared with thermally treated RGO flakes, because the reduction of functional groups not only restores the


Programmed death ligand 1 (PD-L1) expression is an efficient strategy of tumor cells to escape immunological eradiation. However, only little is known about the factors that affect the cellular expression levels. Here we assessed the PD-L1 expression on different breast cancer cell lines under standard in vitro culture conditions and as a function of Epirubicin or Paclitaxel treatment. Moreover, we evaluated the expression in immunodeficient tumor mice as well as in humanized tumor mice . We found highest PD-L1 levels in JIMT-1 and MDA-MB-231 cells. Epirubicin treatment caused a decrease and Paclitaxel treatment an increased PD-L1 expression in MDA-MB-231 cells. In addition, we identified nuclear PD-L1 in MDA-MB-231 cells. All in vivo transplanted breast cancer cell lines downregulated PD-L1 expression compared to their in vitro counterpart. Neither the gene copy number nor the presence of human immune system in humanized tumor mice had an effect on the PD-L1 content. We demonstrate that the degree of PD-L1 expression amongst breast cancer cell lines varies considerably. In addition, cytotoxic treatments and other extrinsic parameters differentially affect the expression. Hence, further investigations including in vivo evaluations are necessary to understand PD-L1 regulation for advanced breast cancer stratification. Accumulating evidences suggest a complex tumor-immune-cell interaction in triple-negative and Her2-positive breast cancer (BC) that is caused by a pronounced immunogenicity of these subentities. The extent of an immune response, reflected by the amount of immune cell infiltration, determines BC prognosis ,2. The tA number of immune modulating approaches are currently being tested in clinical trials in order to neutralize the immune escape by tumor cells. A prominent target that contributes to tumor cell evasion is the immune checkpoint programmed death ligand 1 (PD-L1), also referred to as B7-H1 or CD274. If expressed by tumor cells ,6,7,8. AHere we analyzed the constitutive PD-L1 expression and the cellular localization in subtype-specific BC cell lines as well as the cellular distribution of ER-positive/Her2-negative, ER/Her2 double-positive, and triple-negative BC cell lines under standardized conditions in vitro and as a function of cytotoxic treatments . Moreover, we engrafted triple-negative (MDA-MB-231) and Her2-positive  cell lines in immunocompromised and in humanized tumor mice to assess the potential impact human immune cells on the PD-L1 expression in vivo.The constitutive PD-L1 expression of different BC cell lines under standardized culture conditions was analyzed by flow cytometry, immunochemistry, western blotting, and immunofluorescence . Flow cynullscid IL2Rγ (NSG) mice that were transplanted with human BC cell lines  with or without a simultaneous intrahepatic transplantation of CD34+ hematopoietic stem cells. The transplanted mice developed either solid tumors subcutaneously , liver-associated tumors (BT-474), or tumor effusions in the peritoneal cavity (SK-BR-3). Moreover, mice transplanted with CD34+ cells developed a functional human immune system up to 12 weeks post-transplant. In line with the in vitro data, the highest PD-L1 expression was found in MDA-MB-231 and JIMT-1 BC cell line transplanted animals both in the presence or absence of a human immune system  nor humanized tumor mice (HTM). However, BT-474, MDA-MB-231, SK-BR-3, and JIMT-1 BC cell line tumors apparently showed diminished PD-L1 expression in vivo compared to in vitro cultured cells. In addition, the expression pattern of PD-L1 in MDA-MB-231 and JIMT-1 TM and HTM tumor tissues was very heterogeneous and not expressed ubiquitously. The human immune system in HTMs did not apparently affect the PD-L1 expression in vivo.PD-L1 expression was immunohistochemically analyzed in an in vivo model based on NOD-e system . InteresPD-L1 gene copy number TNBC, luminal, and Her2 overexpressing cell lines were analyzed via a PD-L1 specific fluorescent in-situ hybridization (FISH) probe , except HCC-1937 (TNBC), which demonstrated not only an increased PD-L1 gene copy number but also an increased number of centromere 9 (cen9) signals. MDA-MB-231 and BT-474 BC cells showed a reduced PD-L1 gene copy number that is also reflected in a PD-L1/cen9 ratio < 1.0. In contrast, MDA-MB-453 and HCC-1806 BC cells displayed only enhanced cen9 copy numbers  which indicates some degree of polysomy 9 with a simultaneous loss of chromosomal regions . This finding might indicate instability of chromosome 9. An increased PD-L1 gene copy number in JIMT-1 BC cells could not be found, although this cell line showed the highest cell surface PD-L1 protein expression  probe . PD-L1 gpression . Remarkap < 0.001) and 72 h (p < 0.01) (p < 0.01) (We assessed the PD-L1 expression in MDA-MB-231 cells upon treatment with Epirubicin (Epi) or Paclitaxel (Ptx) for 48 or 72 h by flow cytometry and western blotting. Interestingly, Epi treatment significantly decreased PD-L1 expression after 48 ( < 0.01) A. In con < 0.01) A. The PD < 0.01) B. Fracti < 0.01) C.Cell surface as well as total, cytoplasmic, and nuclear PD-L1 protein expression in Her2-positive SK-BR-3 cells was analyzed upon cytotoxic treatments (Epi or Ptx/48 and 72 h). We found no altered treatment induced PD-L1 cell surface expression nor a redistribution of cell surface located PD-L1 into the nucleus in SK-BR-3 cells (Epi and Ptx after 48 and 72 h) A. HoweveTo date, (neo-) adjuvant chemotherapy is the only systemic approach for the treatment of triple-negative breast cancer (TNBC) and (besides receptor targeting) an essential element for handling Her2-positive tumors. However, the rate of acquired (multi drug) resistance is high and severe side effects are inevitable. Hence, novel biomarkers that indicate the expected treatment efficacy or those that are useful therapy targets are needed for both tumor entities. So-called “immune checkpoint” molecules might meet those requirements. The programmed death ligand 1 (PD-L1)/ programmed death receptor 1 (PD-1)-axis, for example, has been shown to efficiently enable the tumor to escape from the immunological tumor defense ,20, howeIn this study, we assessed the constitutive PD-L1 expression as well as the cellular distribution of ER-positive/Her2-negative, ER/Her2-double-positive, and triple-negative breast cancer (BC) cell lines under standardized conditions in vitro and as a function of cytotoxic treatments. Moreover, PD-L1 expression of different BC cell lines was analyzed in an in vivo tumor mouse (TM) and humanized tumor mouse model (HTM). In accordance with other studies ,14,15,21PD-L1 gene dose . This finding is substantiated by our recent study on 103 primary TNBC tissue specimens [PD-L1 gene copy number and an enhanced PD-L1 gene/cen9 ratio was not found. Interestingly, the high PD-L1 expression in BC cell lines  seems not to be determined by the Instead, the PD-L1 expression on tumor cells seems to be regulated by external factors and can be induced by cytokines  released by immune cells, growth factors or hypoxia ,24. TherNevertheless, SK-BR-3 and JIMT-1 based tumors come with downregulated PD-L1 expression in vivo in TM and HTM compared to their in vitro counterpart. It has been reported that seeding tumor cells at low densities causes an increased nuclear PD-L1 expression, suggesting an association of PD-L1 upregulation as a lack of cell-cell contacts . In contIn addition, a correlation between proliferation (Ki-67 expression) and PD-L1 protein levels have been described in glioma , melanomSince (neo-) adjuvant cytotoxic treatment regimens are common, we investigated the effect of an anthracycline and a taxane treatment on PD-L1 expression in vitro. We found PD-L1 downregulated in triple negative MDA-MB-231 cells when exposed to Epirubicin (Epi) (anthracycline) but an upregulation upon exposition to Paclitaxel (Ptx) (taxane). Obviously, respective cytotoxic treatments differentially affect the PD-L1 expression and individual BC cell subtypes might respond in different ways. This observation is in line with other reports found in the literature. For instance, Doxorubicin (an anthracycline) reduced PD-L1 expression in BC cell lines without any alterations of PD-L1 mRNA levels  whereas Another finding of our study is a time-dependent correlation of the taxane treatment on PD-L1 expression. 48 h Ptx treatment caused an increased PD-L1 expression in MDA-MB-231 cells that dropped back after 72 h to the level of untreated cells. In contrast, the Epi induced PD-L1 suppression remained stable even after 48 and 72 h. These findings demonstrate the dynamic and variable expression range of PD-L1 on tumor cells. With respect to the situation in patient’s tumors it is still uncertain to what extent a modification of PD-L1 expression occurs after chemotherapy treatment and if different PD-L1 levels affect the prognosis of BC patients ,16,31.Only few studies demonstrated an emerging role of nuclear PD-L1 expression in circulating tumor cells and its association with short survival rates of colorectal and metastatic prostate cancer patients ,32. TherOverall, we demonstrate here that the PD-L1 expression and the cellular location is affected by multiple parameters such as 3D cell–cell contact, cytotoxic treatments and others. In addition, we showed that cell lines derived from different BC subentities  respond differently to those factors and therefore represent a dynamically regulated PD-L1 phenotype. Moreover, we observed that even within a given cell line, PD-L1 is heterogeneously expressed. The apparent inconsistency in literature regarding PD-L1 expression might partly be explained by the multifactorial and complex PD-L1 regulation. These ex- and intrinsic factors affecting PD-L1 expression suggests an impact on disease outcome. The evaluation of additional biomarkers that contribute to the regulation of PD-L1 on tumor cells might potentially enhance and specify the prognostic value of PD-L1 expression in BC subentities. Nevertheless, the cell-type-specific regulation of PD-L1 requires further mechanistic exploration.BT-474 , SK-BR-3 , MDA-MB-231  and JIMT-1  BC cell lines were used for cell culture experiments and transplantation to generate tumor mice (TM) and humanized tumor mice (HTM). Moreover, HCC-1806 , HCC-1937 , MDA-MB-361 , MDA-MB-453 , MCF-7 , and ZR-75-1  BC cell lines were used. BT-474, SK-BR-3, MDA-MB-231, MCF-7, ZR-75-1, MDA-MB-453 were cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 5% fetal calf serum (FCS) and for MDA-MB-361 the medium was supplemented with 10% FCS. JIMT-1, HCC-1806, and HCC-1937 were cultured in Roswell Park Memorial Institute (RPMI) medium 1640 supplemented with 5% FCS.scid IL2Rγnull (NSG) mice were obtained from Jackson Laboratories and bred and kept in a specialized pathogen-free facility at the University of Regensburg. HTM were generated by irradiating (1 Gy) newborn animals within the first 48 h after delivery. Three hours later the mice were intrahepatically transplanted with 2.5 × 105 human CD34+ cells isolated from umbilical cord blood together with a subcutaneous or intra-hepatic injection of 3 × 106 BT-474, SK-BR-3, MDA-MB-231, and JIMT-1 tumor cells. In addition, cell lines were transplanted subcutaneously or intrahepatic into newborn irradiated animals without human CD34+ cells to generate tumor mice (TM).HTM were generated as described previously ,34,35. IThe local veterinary authorities of the district government of Lower Franconia and Upper Palatinate (Bavarian region) approved all animal work based on national regulations of the German animal protection act and the European guidelines (permission no. 54-2532.1-27/11 (3 November 2011), 54-2532.1-44/13 (5 November 2014), 55.2 DMS-2532-2-260 (15 July 2016)).Cord blood samples were taken based on the approval given by the Ethics Committee of the University of Regensburg (permission no. 11-101-0287 (23 November 2011), 15-101-0057 (22 April 2015)). All patients included in the study provided written informed consent.5 cells/sample and were grown until they reached a confluence of approximately 70%. Cells were treated with 20 ng/mL Epirubicin (Epi)  or 5 ng/mL Paclitaxel (Ptx)  for 48 and 72 h, respectively. Appropriate sublethal concentration of Epi and Ptx was evaluated based on titrations within the range of 0–100 ng/mL. Untreated cells served as control samples to assess the basal PD-L1 expression. The cells were washed, detached from culture flask, counted, and stained with phycoerythrin (PE)-conjugated anti-CD274 antibody . The appropriate mouse immunoglobulin IgG2b,κ PE  was used for each condition  as isotype control. The basal PD-L1 (CD274) expression and the expression after cytotoxic treatment of the different BC cell lines was determined by flow cytometry using a FACSCanto-II flow cytometer run by the Diva software Ver. 7.0 . The cell lines were seeded in T75 flasks at a concentration of 3 × 10Cell suspensions and tissue samples  were fixed in 5% formalin, embedded in paraffin (FFPE), and cut into 1.5 µm slices. Specimens were deparaffinized followed by rehydration in a descending ethanol gradient . Antigen retrieval was done with Tris/EDTA buffer (pH 9) at 121 °C for 5 min using the decloaking chamber. The slides were cooled down for 10 min in a water bath. After blocking endogenous peroxidase , the slides were washed in TBS-T for 5 min and the primary antibody PD-L1  was applied in appropriate dilution (1:200) for 1 h. Afterwards, the sections were washed in TBS-T and the secondary antibody was applied for 30 min at room temperature. The sections were washed again and incubated for 10 min with DAB plus substrate-chromogen solution . The specimens were rinsed with distilled water and counterstained with hematoxylin for 2 min. Then the sections were rinsed in tap water (5 min), distilled water (1 min) and, dehydrated in ascending ethanol gradient . After 2 × 5 min cleaning steps the specimens were cover-slipped with xylene containing mounting medium.® Isotype Control, Cell Signaling, Danvers, MA, USA) were applied in appropriate dilution for overnight incubation at 4 °C. After a 3 × 5 min washing step with PBS, the slides were incubated for 1 h at room temperature in the dark with goat anti-rabbit IgG AlexaFluor 532 secondary antibody (1:500) . The slides were washed again (3 × 5 min) with PBS and were cover-slipped with VECTASHIELD Antifade Mounting Medium with DAPI .MDA-MB-231, BT-474, SK-BR-3 and JIMT-1 BC cell lines were cultured in chamber slides  up to a confluence of 90%. Immunofluorescence staining was performed according to manufactures protocol of the primary anti-PD-L1 antibody . In brief, the medium was removed and the cells were washed with PBS, PBS was discarded, ice-cold 100% methanol was added and incubated for 10 min at −20 °C. Then the slides were rinsed with PBS for 5 min and incubated with blocking buffer for 60 min. The blocking buffer was aspirated afterwards and the primary anti-PD-L1 antibody (1:100)  or the isotype control (1:833)  or 5 ng/mL Ptx  for 48 and 72 h. Untreated cells were used as control samples. In order to assess the basal PD-L1 content, BC cell lines were lysed for total protein analysis in cell-lysis buffer  supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail . Cytoplasmic and nuclear proteins were extracted using NE-PER™ Nuclear and Cytoplasmic Extraction kit according to manufactures protocol . Twenty-five µg protein per lane  and 17 µg protein per lane (nuclear and cytoplasmic) were separated in 7.5% SDS-PAGE under reducing conditions (mercaptoethanol), and plotted onto Polyvinylidene Difluoride (PVDF) membranes. Membranes were blocked with 5% low-fat milk for 2 h and then incubated overnight at 4 °C with anti-PD-L1 antibody  and anti-β-actin , or anti-Rab11  as loading control for total protein. The monoclonal E1L3N anti-PD-L1 antibody is known to visualize a glycosylated (~50 kDa) and a non-glycosylated (~40 kDa) variant of PD-L1. For cytoplasmic fraction anti-α-Tubulin  and for nuclear fraction anti-lamin A/C  were used as loading control and in order to exclude potential cross-contaminations of the fractions. As protein size standard, the precision plus protein Western C marker  was used. Finally, membranes were hybridized with horseradish peroxidase (HRP)-conjugated goat-anti rabbit IgG  and HRP-conjugated horse anti-mouse IgG . The blots were visualized using the chemiluminescent western blotting detection system  and analyzed by ImageQuant LAS 4000 mini imager .centromere 9 (cen9) region was taken as a surrogate for the number of chromosome 9. The dual marker probe enables to interpret a potential gain of PDCD1LG1 either as a PD-L1 gene amplification or to chromosome 9 polysomy. In brief, 3–4 µm thick deparaffinized tissue sections were pretreated in 98 °C 0.01 M Na-Citrate buffer for 30 min and incubated with pepsin  for 5 min at 37 °C. The sections were rinsed with distilled water and dehydrated in ascending ethanol gradient . Hereafter, 10 µL of the original probe were pipetted on each specimen. The slides were then covered by a coverslip and rubber cement (fixogum™) and after a denaturation step of 5 min at 73 °C the slides were incubated over night at 37 °C. Finally, the coverslip was removed, the samples were washed, and nuclear counter stained with 4′,6-diamidino-2-phenylindole (DAPI). Hybridization spots were then visualized by AxioImager-Z1 microscope  and 25 hybridization spots of nonoverlapping nuclei were counted manually on a cell-by-cell basis.Tissue microarrays (TMA) were constructed from FFPE tumor cell pellets. As previously described in detail , TMA secp values of ≤ 0.05 were considered as significant differences.Results are shown as mean ± SEM. For groupwise comparison, a one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test was performed by using the GraphPad Prism 5 software .