This work thus puts forward a practical route for low-cost mass production of various powdery two-dimensional materials.Mass production of high-quality graphene with low cost is the footstone for its widespread practical applications. We present herein a self-limited growth approach for producing graphene powders by a small-methane-flow chemical vapour deposition process on naturally abundant and industrially widely used diatomite (biosilica) substrates. Distinct from the chemically exfoliated graphene, thus-produced biomorphic graphene is highly crystallized with atomic layer-thickness controllability, structural designability and less noncarbon impurities. In particular, the individual graphene microarchitectures preserve a three-dimensional naturally curved surface morphology of original diatom frustules, effectively overcoming the interlayer stacking and hence giving excellent dispersion performance in fabricating solution-processible electrodes. The graphene films derived from as-made graphene powders, compatible with either rod-coating, or inkjet and roll-to-roll printing techniques, exhibit much higher electrical conductivity (∼110,700 S m High-volume, low-cost production of graphene is pivotal for the industrial advance of this 2D material. Here, the authors make use of naturally occurring diatomite as a 3D substrate for graphene growth, obtaining non-planar porous graphene structures after removal of the silica templates. One of the keys for determining the industrial future of graphene material is its mass production technology with high quality and low cost12356789111314Another route to mass production relies on chemical vapour deposition (CVD) techniques, which generate high-quality graphene films891718202 (80–94%) with chemically removable trace amounts of metal oxides and organics. Diatomite has extremely high porosity with light weight, arising from the hierarchical porous structures of individual diatom frustules, which has been widely used as adsorbent, thermal insulating material, filter, catalyst support, decolouring agent, filling material and so on2425An interesting question is whether we can combine the advantages of CVD graphene and chemically exfoliated graphene together, and develop a practical mass production route to high-quality graphene powders with low cost. For this purpose, we paid our attention to the naturally abundant biotemplates for CVD growth of graphene powders. The possible candidate along this consideration is industrially widely used diatomite222 substrates has been well known with good crystalline quality and much less noncarbon elements as compared with the chemically exfoliated graphene powders27293031In this work, we demonstrate the outstanding performance of diatomite as the template for growing hierarchically structured graphene powders by a surface-oxygen-assisted, small-methane-flow CVD route following a self-limited growth mechanism. Graphene growth on planar SiOCoscinodiscus wailesii diatom source for demonstration. An individual Coscinodiscus wailesii diatom frustule is composed of two almost equal valves  fitting together with a girdle (a circular band of silica), and looks like a Petri dishDiatomite has versatile microstructures depending on the diatom source, or say, producing area. Here we use −1(G band) and 2,700 cm−1(2D band), respectively. Remarkably different from the very weak and broad 2D peak of graphite, as well as the hardly observed 2D peak of RGO, the appearance of sharp 2D peak should indicate the formation of thin-layer graphene with less basal plane defects. The presence of weak Raman D band located at 1,350 cm−1 is attributed to the boundary defects of polycrystalline graphene powders with small domain sizes332930CVD growth of graphene on such biosilica templates changed the original white powders to light grey in macroscopic quantity . RemovinPerfect replication of the fine structures from diatom frustules to graphene flakes is demonstrated in −1  micro-spectrum of HBG flakes shows no discernible peaks from 600 to 1,800 cm−1 , in cont−1 , consist−1 . The oxy−1 .2-bonded carbon structureca. 5 even within sufficient growth durations. Therefore, by selecting suitable methane concentrations, the thickness of graphene layers on the 3D biosilica surface can be on-demand controlled, which is different from those of RGO and LPEG materials.In analogy with previously reported growth of graphene via CVD on planar silica-based substrates274 concentration of 2.4, 0.9 and 0.6 vol%, respectively, after transferred onto 300 nm SiO2/Si substrates. Generally, the colour contrast reflects the thickness of graphene layers, with the darker area assigned to the thicker graphene films, though it is complicated here by the 3D hierarchical structures. Within a single HBG flake, the edge region always shows a darker contrast, which is attributed to the construction of densely aligned graphene channels replicated from the edge channel structures of original diatom frustules. Similar effects can be clearly observed in the central area when increasing the CH4 concentration. The dark dots shown in 4 concentration (0.6 vol%), the HBG is mostly monolayer graphene with a 2D/G intensity ratio of ca. 1.8 and a 2D band full width at half maximum of 47 cm−1, well consistent with the single-layer graphene on planar oxide substrates27334 concentrations. Again, the higher Raman intensity contrast in the 2D and G maps at edges indicates densely aligned graphene channels, in accordance with the optical microscopic images. Shown in 4 after transferred onto SiO2/Si substrates. A statistical average height collected on different graphene flakes is ∼1.5 nm  and the RGO powders (420.9 m2 g−1), which is calculated to be 1,137.2 m2 g−1 by the Brunauer–Emmett–Teller surface area analysis  was found to be best one among the tested solvents for dispersing graphene layers into a stable suspension, due to its perfect match of the polar–polar affinity with graphene (the negative free energy of graphene/NMP mixing system)40−1 exhibits blue photoluminescence, which could be attributed to the strongly localized π state of the sp2 clusters by the electric states of sp3 defect matrixIt is known that good dispersion of graphene powders is the premise for various low-cost and high-performance graphene products such as graphene conductive films, ultralight foams and high-concentration solutions. However, it still remains a challenge to inhibit the restacking of pristine graphene due to its strong interfacial van der Waals interactions and large surface area. As envisioned from the 3D textural features of these atomically thin-layered graphene frameworks, the biomorphic graphene powders show an improved specific surface area compared with the diatomite templates . Besides, a time-consuming chemical/physical exfoliation process  is also required to yield monolayer or few-layer graphene sheets for most RGO and LPEG dispersions, in contrast to the fast wet-etching process for HBG powders. Alternatively, high-concentration dispersion (>1 mg ml−1) is also achieved for ethyl cellulose(EC)-stabilized HBG flakes in ethanol/terpineol within a short timescale of about several minutes, which also shows an excellent stability  while the optical transmittance decreases from 91 to 58% at 550 nm. To enhance their conductivity, these graphene films are further immersed into nitric acid (HNO3). After HNO3 treatment, the average sheet resistance of graphene films drops to 0.36 kΩ sq−1 at a transmittance of ∼80% at 550 nm  and LPEG  counterparts564044For transparent electrode applications, the HBG dispersions can be utilized to prepare large-area graphene conductive films on flexible substrates at room temperature. A wire-wound rod-coating technique was applied to these HBG dispersions to prepare a flexible, continuous and thickness-controllable graphene films on transparent mica substrates . An empihickness . Four-prhickness , and theThe electrical resistance over bending was further monitored to evaluate the mechanical durability of HBG/mica film . In one −1, 25 °C) could be directly written on paper and SiO2/Si wafer, well comparable with the commercial inks. The optical and SEM images in 2/Si substrate exhibit stripes of ∼100 μm width with uniform distribution of graphene flakes, where the so-called ‘coffee ring' effect is almost not found. The sheet resistance of thus-printed graphene film reaches a relatively low value of ∼5 kΩ sq−1 after 30 printing repetitions with this graphene ink , presenting a comparable production rate to those of RGO and LPEG powders  and sulphuric acid (1 mol l−1) to remove the organic and metal impurities, respectively. After the recycled filtration and the deionized water cleaning, the biosilica microflakes with different particle sizes were separated from the filtrated diatomite using recycled sedimentation processes in acetone.The diatom frustules were purified from raw diatomite to obtain the ultrapure biosilica microflakes following several steps. First, the as-received diatomite powders (Aldrich) were immersed and stirred overnight in nitric acid (2 mol l2/Ar carrier gas (300 sccm) and methane (2 sccm) for graphene growth. The thickness of graphene layers could be controlled by varying the concentration of methane gas. After the furnace was cooled to room temperature, light grey powder was obtained. The powder was then immersed in a hydrogen fluoride (HF) etching solution (molar ratio of HF: H2O: EtOH is 6.7: 27.8: 5.1) at room temperature (or a 6 mol l−1 NaOH solution at 80 °C) overnight to remove the biosilica templates. After complete rinsing with water and ethanol, the graphene powder was obtained by freeze-drying at −90 °C and reduced pressure (∼1 Pa) for 24 h. For comparison, RGO was reduced from GO with hydrazine hydrate by modified Hummer's methodDiatomite powders were put into a three-zone tube furnace. The tube was pumped to vacuum and rinsed with 300 standard-state cubic centimetre per minute (sccm) of 10 vol% hydrogen-mixed argon gas to remove air before it was heated to 1,000 °C within 40 min at ambient pressure. The samples were then held at this temperature for 100 min under a constant mixed gas flow of H−1 solution of EC in ethanol was first mixed with the graphene powders. After separated by filtration, the graphene/EC sediment was redispersed in ethanol/terpineol solution with volume ratio of 9: 1 by shaking up to form a stable graphene suspension with different concentrations. One millilitre of the graphene dispersions was dropped on the edge of a mica substrate (50 μm in thickness). A Meyer rod  was pulled over the solution to leave a uniform wet film. During the natural drying process, the graphene films were pressed under a 1 kg weight overnight to improve their formability. After drying, the graphene films were heated in Ar/H2 atmosphere at 350 °C for 60 min, and then rinsed with acetone and dried by blowing with nitrogen. The thickness of graphene films after drying (Hdry) can be controlled by adjusting the concentration of HBG dispersion, according to the following equationHdry=HwetCoρ−1, where the thickness of graphene wet films (Hwet) can be regarded as 10% of the wire diameter of the used wire-wound rod, Co is the concentration of the graphene dispersion, and ρ is approximated as 1.8 g cm−3 referring to the density of graphene oxide films−1, the thicknesses of dried graphene films can be calculated to be about 11.1, 13.3, 25.1, 35.6 and 46.8 nm, respectively.An EC-stabilized graphene dispersion was obtained by adding the as-grown graphene powder into ethanol/terpineol solution containing EC  according to Hersam's method2/Si wafers at a substrate temperature of 100 °C.Prior to inkjet printing, the solution of graphene/EC in ethanol/terpineol was bath ultrasonicated for 60 min to yield small graphene fragments, and then filtered through a polycarbonate membrane with 1 μm pores . The filtered solution was concentrated into graphene/EC terpineol dispersion by heating at 120 °C to remove ethanol, yielding a graphene ink at a concentration of ∼0.2 w/v%. The essential requirement of graphene ink compatible with inkjet printing is its viscosity and surface tension. For successful inkjet printing, the graphene terpineol dispersion was diluted with ethanol at different volume ratios ranged from 1:8 to 1:2 to tailor the viscosity and surface tension. An Epson R330 inkjet printer with a T0851 cartridge was used together with graphene ink for printing pictures on A4 paper and narrow lines on SiO−1 was vacuum-filtrated using a polycarbonate membrane with 0.45 μm pores . The naturally dried graphene film was then pressed on the ethylene-vinyl acetate-coated PET substrate with the graphene side in contact with the substrate at ca. 120 °C.The graphene films could be also prepared by vacuum filtration, followed by a roll-to-roll transferred printing process on PET substrate. A volume of 200 ml of graphene ethanol dispersion with a concentration of 40 μg mlα X-Ray source), thermogravimetric (SDT Q600), XRD , ultraviolet-vis spectroscopy (Perkin Elmer Lambda 950) and static contact-angle measurements (OCA-20 contact angle system) were conducted to characterize the materials, respectively. For SEM and TEM characterization, a PMMA-assisted transfer process was taken for samples onto a SiO2/Si substrate (300 nm oxide layer) and a lacey carbon film supported on copper grids, respectively. For STM characterization, the 4H-SiC(0001) substrates were pre-annealed at ca. 900 °C in vacuum to improve conductivity.Optical microscope (Olympus DX51 microscope), atomic force microscope (Vecco Nanoscope IIIa), SEM , TEM , aberration-corrected atomic-resolution TEM , Raman spectroscopy , ultrahigh vacuum-variable temperature-STM , FTIR , XPS  was used to remove the unwanted regions. Thermal evaporation  was performed to deposit the metal electrodes with chromium/gold (5 nm/100 nm) as the contacts, which were found to form ohmic contact through annealing at 350 °C in Ar/H2 photoelectrode on FTO glass , and clipped together as an open cell for measurements. The cell was then filled with a liquid electrolyte composed of 0.1 M anhydrous LiClO4, 0.12 M I2, 1.0 M DMPII and 0.5 M tert-butylpyridine in dehydrated acetonitrile by capillary force. For comparison, a standard platinum-based counter electrode was also used. The photocurrent–voltage (J–V) measurements were taken using a digital source meter  and a solar simulator  under AM 1.5 G spectra with a light power density of 100 mW cm−2.A sandwich-type solar cell was assembled by placing a PET-supported graphene film (counter electrode) on a N719 dye (Solaronix)-sensitized TiOThe data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Chen, K. et al. Growing three-dimensional biomorphic graphene powders using naturally abundant diatomite templates towards high solution processability. Nat. Commun.7, 13440 doi: 10.1038/ncomms13440 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-25, Supplementary Tables 1-5 and Supplementary References


Several neurological disorders such as Alzheimer’s disease and Parkinson’s disease have become a serious impediment to aging people nowadays. One of the efficient methods used to monitor these neurological disorders is the detection of neurotransmitters such as dopamine. Metal materials, such as gold and platinum, are widely used in this electrochemical detection method; however, low sensitivity and linearity at low dopamine concentrations limit the use of these materials. To overcome these limitations, a silver nanoparticle (SNP) modified electrode covered by graphene oxide for the detection of dopamine was newly developed in this study. For the first time, the surface of an indium tin oxide (ITO) electrode was modified using SNPs and graphene oxide sequentially through the electrochemical deposition method. The developed biosensor provided electrochemical signal enhancement at low dopamine concentrations in comparison with previous biosensors. Therefore, our newly developed SNP modified electrode covered by graphene oxide can be used to monitor neurological diseases through electrochemical signal enhancement at low dopamine concentrations. Dopamine is a key molecule in neurotransmissions in the central and peripheral nervous systems. Therefore, dopamine is responsible for several physiological activities such as behavior, memory and movement ,2. An abTo this end, electrochemical dopamine detection techniques have been intensively studied, owing mostly to their simplicity, rapid response time and excellent sensitivity ,9. DopamHerein, we report upon an electrochemical dopamine biosensor wherein graphene oxide-covered silver nanoparticles (SNPs) were utilized as a core material on an indium tin oxide (ITO) coated electrode . MoreoveA silver (I) nitrate particle sample was purchased from Daejung Chemical & Metals . Sodium sulfate powder was purchased from Junsei Chemical . Phosphate buffered saline (PBS)  solution used as the electrolyte in this study and triton X-100 solution were obtained from Sigma-Aldrich . Single layer graphene oxide (500 mg/L) dispersed in water was purchased from Graphene Supermarket . Sylgard  184 silicone elastomer curing agent and Sylgard 184 silicone elastomer base were acquired from Dow Corning  for polydimethylsiloxane (PDMS) preparation. All aqueous solutions were prepared using deionized water (DIW) from a Millipore Milli-Q water purifier operating at a resistance of 18 MΩ/cm.2 gas. PDMS, an adhesive material, was prepared by mixing a Sylgard® 184 silicone elastomer base and a Sylgard® 184 silicone elastomer curing agent at a 10:1 ratio. The fully mixed PDMS was placed in a vacuum chamber to remove bubbles and stored at −4 °C to avoid curing. After PDMS preparation, the bottom of a plastic chamber (20 mm length × 10 mm width × 5 mm height) was dipped into the prepared PDMS and excess PDMS was removed to procure the area of attachment of the plastic chamber onto ITO electrode. This plastic chamber was then attached to the ITO electrode. Following this, the plastic chamber attached the ITO electrode was heated in a 70 °C oven for 30 min to fabricate an electrode chamber with cured PDMS.ITO electrodes (30 mm length × 10 mm width) were cleaned by sonication for 30 min using a triton-X 0.1% solution, DIW and ethanol, sequentially. The washed ITO electrode was then fully dried by NThe purchased silver (I) nitrate was dissolved in DIW to prepare a 10 mM silver nitrate solution. In order to deposit the SNPs onto the ITO electrode electrochemically, the prepared electrode with the attached chamber was filled with 1 mL of 50 mM silver nitrate solution. The silver nitrate solution in the chamber was well pipetted before the voltage was applied. For the electrochemical deposition of the SNPs onto the electrode, a voltage of −1.3 V was applied to the electrode for 5 s. The graphene oxide solution was diluted to 50 mg/mL using DIW before deposition. This prepared graphene oxide solution was sonicated for 30 min in order to be fully dispersed and centrifuged at 13,000 rpm for 15 min. After centrifugation, sodium sulfate was added as an electrolyte with a 0.1 M concentration. Then, 1 mL of the supernatant of the sonicated and centrifuged graphene oxide was added to the chamber of the SNP-deposited electrode. After that, a voltage of −1.6 V was applied to the electrode for 30 s to obtain the electrochemical deposition of graphene oxide on the electrode. The same method of deposition was used for the fabrication of the ITO electrode covered by graphene oxide. After each electrochemical deposition step, washing of the surface of the modified electrode with DIW was carried out. The structure of the modified electrode was confirmed by scanning electron microscopy .−5 A/V, scan rate of 100 mV/s, quiet time of 2 s and sample interval of 1 mV. The range of the applied potential voltage for CV and DPV was from −0.2 V to 0.6 V. Considering the amperometric i-t technique, −0.3 V, 0.1 s and 1.0 × 10−5 A/V were used for the respective initial potential, sampling interval and sensitivity. Chemical neurotransmitters, namely dopamine, uric acid and ascorbic acid, were dissolved in PBS at various concentrations of dopamine and 50 μM of both uric acid and ascorbic acid. Electrochemical experiments on the fabricated SNP modified electrode covered by graphene oxide were performed using a potentiostat  to confirm the electrochemical signal enhancement. CV, DPV and the amperometric i-t method were performed using a three-electrode system composed of the SNP-modified electrode covered by graphene oxide as the working electrode, a platinum (Pt) wire electrode as the counter electrode and a silver/silver chloride (Ag/AgCl) electrode as the reference electrode. The electrochemical buffer solution (PBS solution) was used for the electrochemical measurement. The parameters of this experiment were a sensitivity of 1.0 × 10The SNP modified electrodes covered by graphene oxide were fabricated using various concentrations of silver nitrate solution and graphene oxide solution with an electrochemical deposition technique in order to find the optimal size of SNPs and thickness of graphene oxide. A plastic chamber was used in both the fabrication process and dopamine detection. A constant inner surface area (19.6 mm length × 8.0 mm width) and a constant volume of silver nitrate, graphene oxide and dopamine could be achieved by using the plastic chamber. Since the volume of the solution was fixed, variables relating to the volume change were removed. The size of SNPs increased or decreased in proportion to the amount of time the voltage was applied for. Likewise, the density of the SNPs on the surface becomes more densely packed as the concentration of silver nitrate solution increased. However, at high a concentration of silver nitrate solution, the number of deposited SNP increased. This resulted in a non-uniform surface of the SNP modified electrode resulting in irregular electrochemical signals. For these reasons, the deposition of SNPs was optimized using a 50 mM silver nitrate solution and 5 s of voltage application time. In a similar way, various concentrations of graphene oxide solution and voltage application times were tested to optimize the condition of graphene oxide deposition. The SEM images of the surface of electrode became dimmer as the concentration of graphene oxide solution increased because of the increased amount of graphene oxide deposition. The voltage application time and sodium sulfate used as an electrolyte for graphene oxide deposition affected the thickness of the graphene oxide. Additionally, graphene oxide was deposited in a sheet structure and if the thickness was too large, the SNP surface would be covered to a great extent. This resulted in a decrease in the electrochemical signal of dopamine and therefore an optimized condition was required. As the concentration of the graphene oxide solution and the voltage application time increased, the thickness of the graphene oxide sheet increased similarly to the relationship between the size of the SNPs and the concentration of silver nitrate and voltage application time. From these results, the condition for optimal graphene oxide deposition was achieved using a 50 mg/mL graphene oxide solution, 30 s of voltage application time and a 0.1 M concentration of sodium sulfate. −1) and G (1580 cm−1) peaks were observed for the ITO covered by graphene oxide and SNP modified electrode covered by graphene oxide, while the bare ITO electrode possessed no peak. The G band was caused by vibration of the SP2-carbon system and the D band was caused by a structural deficit of the graphene oxide. From these results, the presence of the graphene oxide film on the SNP modified electrode was confirmed. Therefore, it could be confirmed that the surface of the ITO electrode, SNP modified electrode, ITO electrode covered by graphene oxide and SNP modified electrode covered by graphene oxide had been successfully fabricated.The morphology of the electrode is shown in pa) was used throughout the experiment. pa values provided by the various electrodes. The electrochemical property of electrode was enhanced compared to the ITO electrode, SNP modified electrode and ITO electrode covered by graphene oxide. This enhancement of the electrochemical peak current was induced by the properties of the SNP and graphene oxide [The enhancement of electrochemical signal was confirmed by comparison among the ITO electrode, SNP modified electrode, ITO electrode covered by graphene oxide and SNP modified electrode covered by graphene oxide. The concentration of dopamine was fixed at 50 μM for comparison. Dopamine was detected at the oxidation peak (0.276 V) by CV. The detection of dopamine was achieved by the adsorption and oxidation of dopamine to the electrode surface. This oxidation of dopamine is an irreversible electrochemical reaction. An oxide layer was thought to be formed when the electrolytic solution was cycled. Since oxide layers increase the adsorption of cationic species, the electrostatic force between the electrode surface and dopamine was increased by the formed oxide layer. Additionally, the achieved electrochemical signal could be unstable when the electrode surface was modified; however, the fabricated electrode surface was constant due to the use of the plastic chamber. For the quantitative assay, an anodic current peak  of three measurements. The detection limit of this electrode was more sensitive and comparable to that of other electrodes used for dopamine detection, as shown in The amperometric i-t technique was performed in order to determine the detecting performance of the SNP modified electrode covered by graphene oxide. To verify the detection limit of the SNP modified electrode covered by graphene oxide, the amperometric i-t technique was conducted with the addition of dopamine at various concentrations . As shown in Uric acid and ascorbic acid coexist with dopamine in the body and demonstrate similar electrochemical properties to those of dopamine ,23. ThusIn this study, an electrochemical biosensor based on electrochemically deposited SNP and graphene oxide was fabricated for use in dopamine detection with an enhanced electrochemical signal, high selectivity and high sensitivity. To achieve the electrochemical signal enhancement, for the first time, SNP was electrochemically deposited onto the ITO electrode followed by the electrochemical deposition of graphene oxide. Compared with previous methods that simply mix SNPs and graphene oxide and deposit the SNPs on graphene oxide, this newly proposed method was attempted. The surfaces of modified electrodes were confirmed by SEM. The electrochemical signal enhancement of the modified electrodes was confirmed by CV. With the deposition of graphene oxide on the SNP surface, the SNP modified electrode covered by graphene oxide showed a much higher enhancement of the electrochemical signal than the SNP- or graphene oxide-modified electrodes. The high selectivity of this biosensor was also confirmed by DPV and an amperometric i-t technique. Dopamine in concentrations ranging from 10 μM to 100 μM was successfully detected in the presence of 50 μM of uric acid and 50 μM of ascorbic acid. The amperometric i-t dopamine detection showed high selectivity towards dopamine. Lastly, the high sensitivity of this electrode was confirmed by the amperometric i-t technique from dopamine concentrations of 0.1 μM to 100 μM as expressing linearity and 0.2 μM as the detection limit. In conclusion, our newly developed biosensor could provide a method for the fabrication of a sensing platform with an enhanced electrochemical signal, high selectivity and high sensitivity in order to detect dopamine in various fields.


We use a split top gate to induce doping of opposite signs in different parts of a graphene field-effect transistor, thereby effectively forming a graphene thermocouple. The thermocouple is sensitive to the electronic temperature in graphene, which can be several hundred kelvin higher than the ambient one at sufficiently high bias current. Combined with the high thermoelectric power of graphene, this allows for i) simple measurements of the electronic temperature and ii) building thermoelectric radiation detectors. A simple prototype graphene thermoelectric detector shows a temperature-independent optical responsivity of around 400 V/W at 94 GHz at temperatures of 4–50 K. S (or thermopower (TEP)) is large, reaching values around 100 µV/K ~ kB/e3. This raises the possibility of a new type of radiation detector based on this effect. Indeed, several studies have indicated TEP as a possible mechanism for the way that graphene devices respond to light irradiation9, for example. However, a number of other explanations are also possible, such as the photovoltaic effect. In addition, placing metal electrodes near graphene provides effective heat reservoirs for non-equilibrium electrons in the graphene, making the TEP effect difficult to detect. Indeed, such heat reservoirs wash out the graphene/metal electronic temperature difference, thereby nullifying the TEP voltage despite the different Seebeck coefficients of the graphene and the metal. Only when the light spot overlaps with both the graphene and its electrode does the corresponding voltage increase significantly9. However, in specially designed experiments with dual-gated graphene, the TEP effects in graphene become much clearer7. Nevertheless, the relatively high transparency of graphene is detrimental to effective light detection.In graphene, the Seebeck coefficient 10. The electronic temperature Te can be assessed by measuring the shot noise, for example. This measurement technique is based on first principles and is straightforward. Several such experiments have shown unambiguously that the electronic temperature can reach several hundred degrees at a high bias current10, which was identified as a working mechanism for the intrinsic photo response in graphene6. However, the technique requires dedicated setups with low-noise microwave amplifiers and often cryogenic temperatures.The electron–phonon coupling in graphene is weak, which can result in significantly different effective temperatures of the electronic and bosonic subsystems when the former is pushed out of equilibrium by a current flowing through the grapheneTe is a very steep function of the Joule power dissipated in graphene, especially at small bias11. Being able to measure small changes in Te accurately by shot-noise measurements would be beneficial for building bolometric radiation detectors. The ultimately small volume of graphene would allow for extremely fast bolometric devices, very likely outperforming the state-of-the-art hot-electron bolometers. Unfortunately, however, this technique does not seem to be particular suitable for measuring small changes in the electronic temperature.The electronic temperature Te due to the bias current or external radiation. We make a split top gate to induce doping of different signs (p- or n-) in two halves of a graphene strip along the direction perpendicular to the current (transverse direction). Any contribution from the longitudinal resistance is largely excluded in this configuration. We also test a simple prototype bolometer by patterning a bow-tie antenna in place of the current-injection contacts. The bolometer shows a clear response to radiation at 94 GHz at both room temperature and 4–50 K, with an estimated optical responsivity of around 400 V/W in the latter case. The noise equivalent power is 20–30 pW/Hz0.5 when using an ordinary lock-in measurement system.Herein, we use the large TEP of graphene to detect a rise in 12. The encapsulation offers long-term stability of the device characteristics and is convenient for making low-ohmic edge contacts to graphene. Figure We make our devices using exfoliated graphene encapsulated in Parylene N (see Methods)V⊥ = 0) in zero magnetic field. The electrostatic doping from the back gate and split top gate allows a p-n junction to form in the middle of the device where the temperature rises because of Joule heating, which is proportional to the square of the current I. Then, because the p- and n parts of graphene have different Seebeck coefficients, the perpendicular voltage becomes non-zero. Because of its thermal origin, V⊥ must be an even function of I; this discriminates a TEP-contribution to V⊥ from those from other sources, such as an inhomogeneous current distribution in the sample.At zero gate voltages and for ideal geometry, we expect no voltage in the transverse direction  and present the results in Fig. V⊥ dependence at T = 4 K and 292 K and at the constant gate voltages Vg1 = −Vg2 = 1 V, which are applied to the two halves of the split top gate; Vbg is the back-gate voltage. We note that the plots corresponding to T = 4 K and 292 K look almost the same, that is V⊥ does not depend on temperature. Moreover, V⊥ is largely positive for both negative and positive I. This confirms unambiguously that the TEP effect is responsible for the nonlinear character of the V⊥(I) dependence at constant Vbg. The relatively small negative values seen in Fig. V⊥ are located at noticeably different (i.e. asymmetric) Vbg. We explain this asymmetry by a self-gating effect when the longitudinal voltage V|| due to a relatively high bias current becomes comparable with the gate voltages. Figure We trace Vg1, Vg, Vbg, IVg1, Vg, Vbg, Ip-n junction crossing the graphene width between the electrodes. The transverse-voltage electrodes can be removed altogether. In this simplified case, the voltage will contain both a DC component from the increased electronic temperature giving rise to a TEP signal and an AC component from the current, which is easy to filter out. Such experiments are under way.The increased electronic temperature of graphene when electrical current flows through it and the easiness of TEP readout are both suited to making radiation detectors. What is needed is to attach an antenna structure to convert incoming radiation into electrical current. Figure V⊥ resulting from irradiating the sample with microwaves. We use a bow-tie antenna to receive the microwave power and convert it to an AC current in the graphene in the middle part of the structure. Similar to Joule heating by a DC current, the idea is that this AC current will heat up the electronic subsystem in the graphene. We then detect the increased Te by using a graphene thermocouple formed by applying voltages of opposite polarity to the split gate. There is no additional current bias in this case.For the second sample see Fig. , we deteVav = (Vg1 + Vg2)/2 at different temperatures below 100 K and for the two polarities of dVg = Vg1 − Vg2 =  ±2 V. The extrema of the response are near the charge-neutrality point at Vav = 1.6 V. When dVg changes sign, the response signal does the same, indicating that the signal has a thermoelectric origin.Figure 2. At about 100 K and above, the signal starts to decrease with temperature down to only roughly 10 µV at room temperature. A clear hysteresis becomes visible when sweeping Vav in time. Frozen out at low temperature, the charge carriers in the silicon substrate gradually thaw at temperatures above 100 K and start screening the graphene from the microwave radiation. Clearly, one should use more insulating substrates to allow the sensors to operate at room temperature.Note that the signal has only a weak temperature dependence over 4–50 K, which is consistent with our DC and low-frequency AC measurements  and a maximum signal value of around 400 µV, we obtain the responsivity Tp and Te, respectively. We use the same sample geometry and materials as in the experiments. We also take into account self gating and two complementary thermoelectric effects (see Methods and supplementary information for assumptions and other details).We model our sensors numerically by solving coupled heat-diffusion equations corresponding to phonon and electron systems with temperatures 10. The hottest part of the graphene is not in the centre of the sample, suggesting that there is room to improve the sample geometry to maximize the TEP voltage. The Peltier effect leads to noticeable spots inside the graphene that are either colder or hotter than the nearby regions near the vertical edges of the top gate  is decreasing when the temperature is lowered2, the TEP signal stays nearly constant at high power, in agreement with the experimental observations  (see supplementary information for more details).We present the calculated temperature distributions in Fig. ate Fig. . In Fig.see Figs . We expl11. Correspondingly, the TEP voltages are large, reaching 100 mV and being easy to measure. At high current, these voltages are nearly the same for all bath temperatures, indicating that the electronic temperature is very high over a wide range of temperature.Figure NEP) by assuming the following experimentally verified parameters. The Seebeck coefficient of graphene is S(T) ~ 100 (T/300[K]) µV/K2. The radiation power P raises the electron temperature Te above the bath (phonon) temperature T0 in accordance with P = β (Te4 – T04), where β ≈ (0.4–2) mW/(m2K4) for low temperature and small power11. Assuming further that the TEP-voltage measurements are limited by 1/f noise in graphene, namely NSD ~ 0.1 µV/Hz0.5 13, we obtain the temperature sensitivity of the graphene thermocouple as δT ≈ NSD/S(T). Then, NEP(T0) ≈ 4βT03δT ≈ 4βT03NSD/S(T) ≈ (40–200) (T0/300)2 pW/(µm2Hz0.5), where we used a linear expansion for small δT = Te−T0 ≪ T0 in the limit of fading power. Counter-intuitively but in qualitative agreement with our experiments, this simple estimation shows that the detector should be more sensitive at low temperature despite the decrease of S. It also shows that the sensitivity is inversely proportional to graphene area. Our experimental estimation of NEP ≈ 25 pW/Hz0.5 (see ‘Response to radiation’ above) could be an order of magnitude lower for a reasonably smaller sample. At room temperature, the power law of electron cooling is different, namely P = α (Te3 – T03), assuming the so-called super-collision cooling10, where α ≈ 0.5 W/(K3m2). A similar estimation of NEP for this case gives NEP(T0) ≈ 3αT02δT ≈ 130 (T0/300) pW/(µm2Hz0.5).A beneficially large TEP in graphene at room temperature allows for a not-so-common readout mechanism in our radiation detectors. Naturally, TEP decreases with decreasing temperature, but the high level of electron heating in graphene at all temperatures more than compensates for this decrease. Indeed, let us make a simple estimation of the minimum detectable power in a 1-Hz band  or narrow-band semiconducting bolometers (>10 pW/Hz0.5) 14. However, the present device is expected to be much faster, with a response rate higher than 200 GHz15. If the noise is limited not by 1/f noise but by the lower Johnson noise, we can expect NEP values that are another order of magnitude better for graphene detectors with TEP readout. Johnson noise is likely to be more relevant for a thermoelectric open circuit with no bias current.A value of We induced inhomogeneous doping in our graphene field-effect transistors to increase the thermoelectric response due to Joule heating by DC or AC currents flowing in the graphene, thereby making the electronic temperature easily accessible. The high thermoelectric power of graphene at room temperature allows for i) simple measurements of the electronic temperature and ii) building radiation detectors with tunable characteristics and low-noise readout. We demonstrated the operation of such a detector by exposing it to microwaves.2/Si. A good optical contrast for the graphene dictates the chosen thicknesses. Doped Si at the bottom of this multilayer ‘sandwich’ serves as a back gate. For radiation-detection experiments, we use undoped Si to decrease microwave losses. At room temperature, the electrical conductivity of the undoped Si is still sufficiently high to be able to use it as a gate electrode. The charge-neutrality point in the majority of our devices lies not far from zero doping because Parylene N is non-polar and hydrophobic12. Typically, the back-gate voltages needed to reach the charge-neutrality point are less than 10 V for the thickness of dielectric layers given above.We first exfoliate graphene with the help of sticky tape and lay it down onto 150 nm/90 nm/0.3 mm of Parylene/SiOµ of the charge carriers estimated from the transfer curves of similar devices but with additional electrodes for measuring the longitudinal resistance is 0.5–1 m2/(V s) .We use e-beam lithography to pattern the graphene structures and their metal contacts after first covering the graphene with the top 90-nm-thick Parylene layer, thereby fully encapsulating the graphene. Oxygen-plasma etching of Parylene and graphene exposes the graphene edges. Lift-off patterning of the Cr/Pd/Au metallization allows these edges to be contacted and the electrodes to be shaped. Finally, we pattern a split top gate making sure it does not contact the graphene edges by covering them with an extra layer of Parylene. The mobility For the experiments with microwaves, we glue the sample back-to-back onto a Si hyper-hemispherical lens of 10 mm in diameter and place it into a gas-flow cryostat (Janis PT950) with optical windows made of z-cut quartz. We use a Gunn-diode oscillator at 94 GHz and 30 mW with a conical-horn antenna to generate and direct the microwaves to the sample through the windows. A doped Si wafer in front of the cryostat attenuates the power down to roughly 100 µW. Further attenuation comes from the reflection losses at the window surfaces, from the Si lens and because of geometrical factors. In total, we estimate the optical power reaching the sample to be roughly 1 µW.0.5 at 59 Hz with the sample connected but the cryostat compressor switched off.A mechanical chopper (SR540) modulates the microwave beam periodically at a frequency of 59 Hz. The periodic response signal from the sample is amplified by an instrumentation amplifier  and is measured by a lock-in amplifier (SR850) using a 24-dB/Octave filter and a time constant of 0.1–0.3 s . The overall noise floor of the measurement system is roughly 10 nV/Hzσ = μ [e2n02 + (CsVg)2]0.5, where e, n0 (= 3 × 1011 cm−2), Cs/e (= 8.14 × 1010 cm−2 V−1) and Vg are the electron charge, the residual charge density in graphene, the gate capacitance and the gate voltage, respectively. We assume the conductance and the phonon thermal conductivity kp = 600 W/(K m) to be independent of temperature. The phonon energy is dissipated to the Si substrate through the Parylene layer. The weak coupling between the phonon and electron systems depends on the phonon temperature T0 and the Bloch–Grüneisen temperature TBG. For T0 > TBG, the electron cooling by phonons is cubic in temperature, namely Pp-e = α (Te3 − T03), whereas for T0 < TBG the cooling power is Pp-e = β (Te4 − T04), with α ≈ 0.5 W/(K3 m2) and β ≈ 2 × 10−3 W/(K4 m2)10. The electron thermal conductivity is taken as ke = γ(Te/T0)1.6, where γ ≈ 6.8 W/(K m)16. The Seebeck coefficient for graphene is simulated by the Mott equation, namely S(T) = zT[∂ln(σ(E))/∂E], with the coefficient z to adjust the maximum of |S| to the experimentally observed 100 µV/K at T = 300 K2. Although questionable for graphene, the Mott equation reproduces the experimental dependence of S on doping and temperature qualitatively well2. The Seebeck, Peltier and self-gating effects are included in the calculations (see supplementary information for more details).We carry out computer simulations of the temperature distribution and calculate the TEP voltage with the help of the COMSOL 5.2 software and assuming the same sample geometry as for the experiments see Fig. . We perfThe datasets generated during and/or analysed during the current study are are included in this published article (and its Supplementary Information files) or available from the corresponding author on reasonable request.Thermoelectric effects in graphene at high bias current and under microwave irradiation (Supplementary Information)


Idiopathic pulmonary fibrosis (IPF) identifies a specific lung disorder characterized by chronic, progressive fibrosing interstitial pneumonia of unknown etiology, which lacks effective treatment. According to the current pathogenic perspective, the aberrant proliferative events in IPF resemble those occurring during malignant transformation.druggable in IPF. One example of an RTK—the MET proto-oncogene—is a key regulator of invasive growth. This physiological genetic program supports embryonic development and post-natal organ regeneration, as well as cooperating in the evolution of cancer metastasis when aberrantly activated. Growing evidence sustains that MET activation may collaborate in maintaining tissue plasticity and the regenerative potential that characterizes IPF.Receptor tyrosine kinases (RTK) are known to be key players in cancer onset and progression. It has been demonstrated that RTK expression is sometimes also altered and even The present work aims to elucidate—by applying the logic of simplicity—the bio-molecular mechanisms involved in MET activation in IPF. This clarification is crucial to accurately design MET blockade strategies within a fully personalized approach to IPF. The mede in IPF , 5. The e in IPF . The METe in IPF , which iMET gene, located on chromosome 7q31, encodes for the TK receptor for ‘Scatter Factor’ or Hepatocyte Growth Factor (HGF), which detects adverse micro-environmental conditions and drives cell invasion and metastasis through the transcriptional activation of the invasive growth signature, a genetic program also defined as the epithelial-mesenchymal transition (EMT) [anoikis and angiogenesis. MET is now a prominent target in cancer therapy, with several compounds in active clinical development  structural alteration, such as: point mutations which cause increased kinase activity; oncogene rearrangement ; abnormal post-translational processing resulting in a constitutively active molecule exposed on the cell membrane; impaired down-regulation generally due to mutations that prevent binding of the Cbl ubiquitin ligase which is responsible for MET ubiquitination and endocytosis thus leading to increased receptor expression at cell surface and enhanced signal transduction. In addition, naturally truncated and active MET receptors have been detected in malignant human musculoskeletal tumors; (iii) HGF-independent autocrine-paracrine activation. In these contexts, paracrine activation—typical of physiological conditions—can become pathological in the presence of abnormal HGF production by mesenchymal cells. Autocrine activation occurs when tumor cells aberrantly express both HGF and its receptor, as observed in rhabdomyosarcomas, gliomas, carcinomas of thyroid, breast and lung cancers, (iv) HGF-independent mechanisms. Moreover MET phosphorylation can also occur through transactivation by other membrane receptors, including adhesive receptors such as CD44, integrins, signal transducing receptors  [for a detailed review see H. Pylori, associated with gastro-esophageal reflux disease which, in turn, is known to be implicated in the development of IPF, giving rise to recurrent lung insult [The MET proto-oncogene is a key regulator of the genetic program known as invasive growth. The on (EMT) . The latg insult , 15. Notg insult , 17. Rapg insult , 19. A gg insult –23 in seg insult –26. In cg insult , 28. Altg insult , 29, derfibroblast foci (FF) defined as aggregates of actively proliferating fibroblasts and myofibroblasts. Activated fibroblasts express α-SMA (smooth muscle actin), accounting for to term “myofibroblasts”. In addition they secrete increased levels of ECM-degrading proteases , facilitating increased ECM turnover and altered ECM deposition; they also secrete growth factors  which can induce proliferative signals within adjacent epithelial cells. Moreover activated fibroblasts behave as modulators of the immune response following tissue injury by secreting cytokines (e.g. IL-1) and chemokines (e.g. MCP1) [MET coding sequence has been reported; MET mutations have been biologically associated to the observed transformed phenotype [IPF is a proliferative disorder affecting the lungs, characterized by aberrant deposition of ECM and consequent remodeling associated with the activation of fibroblasts as a response to still unknown injuries. IPF diagnosis is confirmed by histological identification of the usual interstitial pneumonia (UIP) pattern on surgical  biopsies, together with detection at high resolution (HR) computerized tomography (CT) scan of bibasilar reticular abnormalities (honeycombing pattern) with minimal or absent ground-glass opacities , 102. Thg. MCP1) –107. Actg. MCP1) . Throughg. MCP1) . The acqg. MCP1) . Thus emg. MCP1) , 112. HGg. MCP1) , 114. Mog. MCP1)  but can g. MCP1) , known ag. MCP1) . Sometimhenotype . The abohenotype , 120. Eahenotype . MET-drihenotype , 122–125henotype . These fhenotype –129. Whehenotype . Moreovehenotype , 132. Grhenotype –136. As pluralitas non est ponenda sine necessitate  can be applied to correctly understand the role of MET-driven invasive growth at disease onset. IPF resembles cancer in many MET-associated behaviors, such as invasive phenotype and pro-coagulant status. However dynamics of malignant divergent clonal selective pressure and heterogeneity clearly differ from those occurring in IPF and impact on the biological significance of MET activation. The RTK MET is phosphorylated in myofibroblasts in FF: in a context-specific regulation of its expression, MET might become a functional marker of IPF and an actionable target. The cytogenetic heterogeneity, that is a hallmark of FF, can be exploited for therapeutic purposes and already commercially available MET-inhibitors can be tested to interfere with IPF progression. MET-mediated events in IPF rely on qualitative differences among physiological signals, whereas no driver genetic lesions, causally implicated in the disease can be clearly demonstrated. Thus MET blockage falls among those therapeutic strategies aimed to impair the “aberrant recapitulation of developmental programs” as Selma and coll. already defined IPF [In a complex and heterogeneous setting, which applies to IPF, the principle of ined IPF . A dynamined IPF , 136. Thined IPF , 139. Wnined IPF , 140. Alined IPF –144. Conined IPF –149. In ined IPF –153. Theined IPF , 154. Mained IPF . In partined IPF –159. HGFined IPF  and ERK-ined IPF , 162. Neined IPF . Anotherined IPF –166. It ined IPF . More imined IPF . It is pined IPF  to gain The molecular pathways involved in the metastatic process in cancer are shared with IPF. Among them the MET-driven invasive growth program plays a crucial role. As discussed above, MET activation governs a number of physiological and pathological processes that modulate dynamic changes and plasticity of tissues. If in cancer MET activation enables cells to overcome damage induced by targeted agents and ionizing radiation, there is enough evidence to sustain that in IPF the versatility of the MET-mediated biological responses may promote tissue remodeling by integrating growth, survival and migration cues in response to abnormal environmental stimuli or cell-autonomous perturbations in absence of addiction phenomena. More likely, MET expression in myofibroblasts behaves as in cancer stem cells, where it sustains the inherent self-renewing, self-preserving and invasive growth phenotype , 19. The


To the editor: We greatly appreciate the editorial by Penttinen and Friede summarising the data regarding recent observations in the United States (US) of decreased effectiveness of the influenza A(H1N1)pdm09 strains  included in live attenuated influenza vaccines (LAIV) [luenza AHN1pdm09 sBased on evidence presently available to us, we believe that reduced replicative fitness of the A/California and A/Bolivia (H1N1)pdm09 LAIV strains is the most probable root cause for the reduced vaccine effectiveness (VE). From 2010/11 through 2013/14, LAIV VE in children aged 2–17 years against matched A(H3N2) and B strains has been comparable to that observed with IIV . LaboratIn the context of reduced replicative fitness, vaccine–virus interference may have contributed to the observed reduced VE. However, vaccine–virus interference specific to the quadrivalent formulation appears to be an unlikely root cause of the reduced VE with LAIV. LAIV3 demonstrated reduced VE against A(H1N1)pdm09 in 2010/11 in the US . AdditioBecause rates of vaccine coverage in the US have historically been higher compared with European countries, questions have been raised regarding the role of prior vaccination in the reduced AH1N1)pdm09 effectiveness N1pdm09 e-10. No sIn 2013/14, with LAIV4 containing the A/California strain, a statistically significant correlation was observed between reduced LAIV VE against (H1N1)pdm09 viruses and higher outdoor temperatures during LAIV lot unloading at US distributors . In laboWe have initiated a multifaceted scientific investigation into the causes of the recently observed reduced effectiveness of LAIV, with the goal of identifying a more effective A(H1N1)pdm09 LAIV strain for potential inclusion in the 2017/18 LAIV formulation. All potential hypotheses continue to be evaluated. We welcome the input and support of the multiple stakeholders involved, including national public health agencies, the World Health Organization, and additional external scientific experts, as we work together to ensure that VE of LAIV is improved in future influenza seasons.


Porcine parvovirus type 1 is a major causative agent of swine reproductive failure. During the past decade, several new parvoviruses have been discovered in pigs. Porcine parvovirus type 6 (PPV6), recently identified, has been reported in pigs in China and in the USA while the PPV6 status in the European pig population remains undetermined. In the present study, PPV6 DNA was identified in serum samples collected from domestic pigs in Poland. In investigated herds, the prevalence of PPV6 was 14.9 % (15/101 samples). Sequencing was conducted, and 11 nearly complete PPV6 genomes were obtained. Phylogenetic analysis indicated that PPV6 sequences cluster into four distinct groups, and the Polish PPV6 strains from three individual farms were present in three of these four groups. In addition, the Polish PPV6 strain P15-1 was identified as a putative recombination of an ORF1 from US stains and an ORF2 from Chinese strains. This is the first identification of PPV6 in Europe, and this finding will encourage future epidemiological studies on parvoviruses in European pigs.The online version of this article (doi:10.1007/s11262-016-1386-y) contains supplementary material, which is available to authorized users. Parvoviridae consists of two subfamilies: the Parvovirinae infecting vertebrates and the Densovirinae infecting arthropods. The subfamily Parvovirinae can be further divided into eight genera [Protoparvovirus, Bocaparvovirus, Copiparvovirus, and Tetraparvovirus contain viruses that infect pigs.Parvoviruses are small, non-enveloped DNA viruses with a single-stranded linear genome of approximately 4.0–6.3 kb in size containing two to three main open reading frames (ORFs) , 2. The Protoparvovirus, the emerging PPV species belong to the genera Tetraparvovirus  and Copiparvovirus  [Porcine parvovirus 1 (PPV1) is a well-known pathogen in pigs and frequently associated with reproductive failure in breeding herds, resulting in widespread breeding herd vaccination in an effort to control this virus . PPV1 waPPV2, PPV and CopiParvovirinae and is most closely related to PPV4 [PPV6 shares only 20.5–42.6 % nucleotide similarity with other members of the subfamily  to PPV4 . The disn = 101) were collected from 2- to 18-week-old commercial cross-bred pigs located on three commercial Polish pig farms designated as U, K, and P. The herds ranged in size from 600 to 1000 pigs. Total nucleic acids were extracted from serum samples using the MagMAX™ Viral RNA Extraction Kit (Thermo Scientific) on an automated extraction system (Thermo Scientific Kingfisher Flex) according to the manufacturer’s instructions. The extracted samples were stored at −20 °C until testing.Serum samples  within the region of the detection primers were designed using Primer Express software . The PCRs were carried out in 96-well plates, with each reaction containing a total volume of 25 μl, 12.5 μl of the TaqMan® Universal PCR Master Mix (Applied Biosystems®), 2.5 μl of the sample or standard DNA, 1 μl of 10 μM of each of the two primers, 0.5 μl of 10 μM probe, and 7.5 μl nuclease-free water. Amplification reactions were performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems®) under universal conditions: 2 min at 50 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. Samples with no cycle threshold (CT) at 37 cycles were considered negative. The specificity of the probe was confirmed by BLAST analysis and using DNA samples positive for other DNA viruses including PPV1 through PPV5, porcine circoviruses 1 and 2, and Torque teno sus virus. The sensitivity of the PPV6 real-time PCR assay was determined by testing 10-fold serial dilutions of the PPV6 DNA standard in triplicate. The generated standard curve with a regression value of R2 = 0.989 was linear and showed a reaction efficiency of 114.092. The detection limit was 50 genome copies per reaction with Ct values around 36 in all three replicates. Real-time positive samples were also tested by a conventional PCR assay as previously described [2O under the following thermocycler conditions: denaturation for 30 s at 98 °C followed by 30 cycles of 98 °C for 10 s, 55 °C for 30 s, and 72 °C for 60 s, and a final extension step at 72 °C for 10 min. The PCR products were purified by the PureLink™ Quick Gel Extraction and PCR Purification Combo Kit  according to the manufacturer’s instructions and were sequenced at the Edinburgh Genomics facility, University of Edinburgh, Edinburgh, United Kingdom. The sequences were assembled by the SeqMan program of Lasergene 7.0 software  and compared with other available PPV6 sequences using the BLAST web-based program (http://www.ncbi.nlm.nih.gov/BLAST). PPV6 sequences were then aligned with available reference sequences downloaded from GenBank by Clustal Omega (http://www.ebi.ac.uk/). Phylogenetic trees were inferred by maximum-likelihood (ML) method (amino acid sequences) or neighbor-joining method (nucleotide sequences) implemented in MEGA v7.0 [Initial detection of PPV6 DNA in the sample set was done by a real-time PCR developed based on the alignments of PPV6 available in the GenBank. A pair of primers  targeting 154 bp in ORF2 and a specific TaqManParvovirinae, all PPV6 strains identified in Poland were closely related to PPV6 reference strains and located in the genus Copiparvovirus together with PPV4 and PPV5  of the samples. The individual PPV6 farm prevalence rate ranged from 3.8 % for farm P to 22.2 % for farm K (Table S2). Eleven of the PCR positive samples were sequenced (Table S2), and the nucleotide sequences of these nearly complete PPV6 genomes were deposited into GenBank under the accession numbers KX384822 (U18-1), KX384814 (U18-4), KX384818 (U18-5), KX384815 U18-7), KX384816 (U18-8), KX384817 (U18-9), KX384819 (K13-4), KX384813 (K13-8), KX384821 (K17-3), KX384820 (K17-10), and KX384823 (P15-1). The complete coding regions (excluding 5′ and 3′ UTR) of the Polish PPV6 strains were 5566 nucleotides (nt) in length and contained two major open reading frames (ORFs). ORF1 (1989 nt) encoded the NS1 protein with 663 amino acids (aa) and ORF2 (3570 nt) encoded the VP1 protein with 1190 aa. Based on amino acid sequences of the NS1 protein which is commonly used to differentiate members of the subfamily , KX38481The complete coding regions of the identified PPV6 strains shared 98.0–99.8 % nucleotide sequence identity with US PPV6 strains and 96.4–98.9 % nucleotide sequence identity with Chinese PPV6 strains. Sequences isolated from the same farm showed a high nucleotide identity (98.6–99.9 %) to each other. The motifs of the calcium binding loop (YXGXR) and the catalytic residues (HDXXY) of PLA2 were found to be conserved among PPV6 strains. Compared with NS1, VP1 resembles the variable protein in the PPV6 genome. The VP1 sequences of the investigated Polish PPV6 strains showed 94.3–100 % nucleotide identity (95.0–100 % amino acid identity) with reference sequences. A previous study indicated that there may be three conserved regions in the PPV6 VP1 protein . HoweverOur findings demonstrate that PPV6 circulates in swine herds in Poland. This study represents the first identification of PPV6 in European pigs, indicating that PPV6 is not restricted to Asia and North America. Phylogenetic analysis suggests that the PPV6 P15-1 strain might be a chimeric virus containing an ORF1 gene from the USA and an ORF2 gene from China. Further studies are ongoing to investigate the epidemiology and evolution of parvovirus in European pigs. To date, the exact relatedness of pathogenesis and occurrence of novel species of PPV strains in pigs remains unknown.Supplementary material 1 (DOCX 13 kb)Below is the link to the electronic supplementary material.


Neural stem cells (NSCs) are special types of cells with the potential for self-renewal and multi-directional differentiation. NSCs are regulated by multiple pathways and pathway related transcription factors during the process of proliferation and differentiation. Numerous studies have shown that the compound medicinal preparations, single herbs, and herb extracts in traditional Chinese medicine (TCM) have specific roles in regulating the proliferation and differentiation of NSCs. In this study, we investigate the markers of NSCs in various stages of differentiation, the related pathways regulating the proliferation and differentiation, and the corresponding transcription factors in the pathways. We also review the influence of TCM on NSC proliferation and differentiation, to facilitate the development of TCM in neural regeneration and neurodegenerative diseases. Both embryonic and adult neural stem cells (NSCs) are widely distributed in the nervous system. Embryonic NSCs are widely distributed in the brain, while adult NSCs are mainly distributed in the subventricular zone (SVZ) of the lateral ventricle wall and the subgranular zone (SGZ) of the hippocampus dentate gyrus , 2. NSCsNSCs can differentiate into different types of neural cells under specific conditions, which can provide new methods of cerebral injury repair and neurological disease treatment. Numerous studies have shown that NSCs have a therapeutic effect on nervous system injuries and degenerative diseases such as Alzheimer’s disease (AD), Parkinson's disease (PD), spinal injury, amyotrophic lateral sclerosis (ALS), vascular dementia, cerebral hemorrhage, and Huntington’s disease -17. HoweTraditional Chinese medicine (TCM) has the general advantages of multi-targets, multi-levels, and multi-paths -21. It cIn this article, we discuss the regulation of NSC proliferation and differentiation by the relevant pathways and the target genes corresponding to the pathways, and review the effects of TCM on the proliferation and differentiation of NSCs, to further develop the study of NSC proliferation and differentiation by TCM.NSCs go through different stages of NSPCs, NPCs, and neuroblasts in the process of proliferation and differentiation into mature neural cells -32. NSCsAn increasing number of novel markers have also been used to identify NSCs. These include homeodomain-only protein X (Hopx) , hairy aMultiple signaling pathways regulate the process of NSCs proliferating and differentiating into mature neurons. These pathways determine the fate of NSCs by regulating the expression and activity of different transcription factors, and many pathways are involved. The Notch, Wnt, bone morphogenetic protein (BMP), and sonic hedgehog (shh) signal pathways have been most studied. Downstream of the signal pathway are usually target genes that can be used as transcription factors to regulate the process of proliferation, differentiation, and migration of NSCs, and different signaling pathways and transcription factors can act synergistically to regulate this process. These are summarized in in vivo and in vitro. Hes1 is mainly expressed in the SVZ. It can maintain the state of NSCs, inhibit their differentiation into neurons, and also have an effect on the maintenance and self-renewal of NSPCs [in vivo and in vitro. Studies have shown that Mash1 promote the differentiation of NSPCs and NPCs [in vivo and in vitro [in vivo. It also plays a specific role in the maintenance of NSPCs [in vivo and in vitro. Ngn2 also controls the balance between the maintenance and differentiation of NSPCs [The Notch gene was originally found by Morgan and colleagues in drosophila in 1917 . The parof NSPCs -87. Hes5of NSPCs -90. Mashand NPCs . NeuroD,in vitro -102. zfpin vitro , 104. Ngof NSPCs , 106. Ngof NSPCs , 108. Feof NSPCs , 110. Heof NSPCs -113. Gsxof NSPCs -116.2+ pathway, and the intracellular pathways that regulate spindle orientation and asymmetric cell division [in vivo and in vitro. Studies have shown that Pax6 can be expressed in both SVZ and OB, and can control the balance between the self-renewal and differentiation of NSCs, and is important in their maintenance, self-renewal, and multi-directional differentiation [in vivo and in vitro. Emx2 mainly affects NPCs through controlling the migration and differentiation of NPCs [in vivo and in vitro. Oct4 is important in the maintenance of pluripotent stem cells, and promotes the proliferation and self-renewal of NSCs [The Wnt signaling pathway is named after its promoter protein Wnt, which is synthesized by the wingless gene of the African drosophila and the proto-oncogene Int1 of the mouse. The four main Wnt signal pathways are the canonical Wnt/β-catenin pathway, the Wnt/polarity pathway, the Wnt/Cadivision . Of thesdivision -120. Thentiation -126. Emx of NPCs -129. Dix of NPCs , 131. Pa of NPCs -137. Oct of NSCs -140. Pro of NSCs , 142.in vivo and in vitro and promote the proliferation of NPCs [The hedgehog gene was first found in Drosophila in 1980, and has three homologous genes: sonic hedgehog (Shh), Indian hedgehog (Ihh), and desert hedgehog (Dhh). They encode Shh, Ihh, and Dhh proteins, respectively -145. The of NPCs -162.in vitro, and promotes the maturation of differentiated cells [Bone morphogenetic protein (BMP) is a type of acidic peptide, and members of the transforming growth factor β (TGF-β) superfamily , 164. BMed cells -175.Numerous studies have shown that TCM has a regulatory effect on NSC proliferation and differentiation. TCM can improve the microenvironment, promote neurogenesis, repair nerve damage, and provide new treatments for cerebral injury and neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and strokes . Here, w+significantly decreased and expression of neurofilament (NF) and glial fibrillary acidic protein (GFAP) significantly increased in the NSPCs treated with Buyang Huanwu Decoction [Experimental studies have found that compound Chinese medicine preparation has an important regulatory role on NSCs proliferation and differentiation. The compound prescriptions mainly include Huoxue Huayu (promoting blood circulation and removing blood stasis) and tonifying kidney recipes, of which Buyang Huanwu Decoction is a classic TCM prescription. This can promote blood circulation and dredge the meridians, and thus often used for the treatment of cerebrovascular disease. Buyang Huanwu is composed of Astragalus membranaceus (Fisch.) Bge (120 g), Angelica sinensis (Oilv.), Diells (10 g), Paeonia lactiflora Pall (10 g), Ligusticumwallichii Franch (10 g), Carthamus tinctorius L (10 g), Semen Persicae (10 g) and Flos carthami (4.5 g). Recent research has demonstrated that Buyang Huanwu Decoction can promote the proliferation and differentiation of NSCs, and improve the expression of growth-associated protein-43 (GAP-43) . It can ecoction .Jiaweisinisan consists of Stellaria dichotoma L. var. lanceolata Bge, Paeonia lactiflora Pall, Citrus reticulata Banco, Poncirus trifoliate (L.) Raf, Lycium barbarum L, Gardenia jasminoides Ellis, Radix Rehmanniae, and Abalone. These are weighed according to the ratio of 1:3:1:3:1:4:6, respectively. This Jiaweisinisan prescription can also promote the proliferation of hippocampal NPCs, and inhibit the apoptosis of glial cells and neurons differentiated from Hippocampal-NPCs .Shengyu decoction, a traditional Chinese medicine, has been used to treat diseases that involve a deficit in “qi” and “blood.” Modified Shengyu decoction (MSD) was designed to treat brain injury after head trauma, according to traditional Chinese medicine theories, and is based on the traditional Shengyu. Four additional herbs are in the MSD: Salvia mil-tiorrhiza Bunge, Commiphora myrrha (Nees) Engl., Acorus calamus L, and Curcuma aromatica Salisb. A study on the treatment of traumatic injury in rats using the Shengyu decoction showed that it could increase the expression of nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), neural cell adhesion molecule (NCAM), and Tenascin-C (TN-C) in the cortex and hippocampus of rats. It can also inhibit the expression of Nogo-A and promote the proliferation of NSCs/NSPCs and their differentiation into neurons .Fuzhisan is a traditional Chinese medicine prescription, composed mainly of four Chinese medicinal herbs: Panax ginseng C. A. Mey, Scutellaria baicalensis Georgi, Acorus gramineus Soland, and Glycyrrhiza uralensis Fisch. Experimental studies have shown that Fuzhisan can promote the proliferation of NSPCs and improve the survival rate of newborn cells .Xiehuo Bushen Decoction consists of Rheum officinale baill, Paeonia suffruticosa Andr, Paeonia lactiflora Pall, Astragalus membranaceus (Fisch.) Bge, Cuscuta Lam, Viscum coloratum (Kom.), and Nakai. It can promote the survival and differentiation of NSCs transplanted into a brain after cerebral hemorrhage. The possible mechanism is that the Xiehuo Bushen Decoction can enhance the expression of interleukin 4 (IL-4) mRNA and down-regulate the expression of interferon-gamma (IFN-gama) mRNA .Polygonummultiflorum Thunberg complex composition-12 (PMC-12), a mixture of four medicinal herbs, includes Polygonum multiflorum Thunb, Radix Polygalae, Rehmannia glutinosa, and Acorus gramineus Soland. PMC-12 was found to promote the proliferation of NSPCs in the hippocampus, increase the survival rate of newborn neurons, and encourage neurogenesis in the hippocampus .Although compounds of Chinese medicine have been found to regulate the proliferation and differentiation of NSCs through experiments, it has not been determined whether one or a combination of the active ingredients influences the proliferation and differentiation of the NSCs, because of the complexity of the active ingredients of the compounds of Chinese medicine. Further experiments are therefore required to establish this.Extensive research has been conducted on Chinese herbs, such as those used for Huoxuehuayu and Qingrejiedu  in the study of NSC proliferation and differentiation.in vitro, and promote the survival and differentiation of NSCs derived from multifunctional stem cells [in vitro experiments to promote the proliferation of NSCs, but the study was conducted together with the herbs of Qingrejiedu [Salvia miltiorrhiza Bge is a common traditional Chinese herb for Huoxuehuayu. It has anti-oxidation and anti-inflammatory functions and is often used to treat nervous system diseases. Studies have shown that Danshen can increase the expression of the nestin significantly, promote the differentiation of induced multifunctional NSCs into neurons em cells . Anothergrejiedu .in vitro. In vitro study shows that this herbal medicine can improve the symptoms of depression in mice models, which was mainly achieved through the machanism of increasing the content of corticosterone and promoting hippocampal precursor cell proliferation [The herbal preparation composed of Scutellariacalensis Georgi, Phellodendron chinense Schneid, and Ligusticumwallichii Franch has been shown to promote the proliferation of NSCs feration .Experiments also showed that the traditional Chinese herb Sambucus williamsii Hance can promote the differentiation of induced pluripotent stem cells (iPSCs) into neurons by up-regulating the expression of tubulin-1 (Tuj1) and nestin, and down-regulating the expression of Oct4 and Sox2 .In recent years, numerous experiments have demonstrated that the extract of Chinese herbal monomer Plays a specific regulatory role in NSC proliferation and differentiation. The current research mainly focuses on the extraction of effective components from the Chinese tonifying herbs HuoxueHuayu and QingreJiedu .Panax ginseng C. A. Mey is an herb used in TCM for tonifying “Qi”. Recent research has shown that it has roles in anti-anxiety, anti-depression, and cognitive function-enhancing, among others. The numerous active ingredients in Panax ginseng C. A. Mey have the effect of nerve protection . Ginsenoin vitro. The relevant mechanism may be the increase of the expression of tubulin and the ratio of tubulin/DAPI [Oleanolic acid extracted from the “Yin”-tonifying Chinese herbal Ligustrum lucidum Ait and stilbene glycoside extracted from Fallopia multiflora (Thunb.) Harald have been found to promote the self-renewal and differentiation of NSCs lin/DAPI .Resveratrol is a polyphenolic compound found in various plants, and is an effective component of the Chinese herb Fructus Mori. Experiments have established that it can promote the survival and proliferation of NSCs. In addition, the expression of Patched-1 (Ptch-1), Smoothened (Smo), Gli-1 protein, and RNA were all up-regulated in resveratrol-treated NSCs , 158.The tortoiseshell cholesterol extracted from Chinemys reevesii (Gray), (+)-Cholesten-3-one, can induce NSCs into dopaminergic neurons through a bone morphogenetic protein (BMP) signal, thus providing a possible new treatment for PD .in vitro, thereby promoting the differentiation of NSCs into astrocytes [Psoralen is an effective component extracted from the Chinese herb , and is representative of kidney-tonifying herbs. Psoralen has been found to increase the expression of glial fibrillary acidic protein (GFAP) in NSCs trocytes . Icariintrocytes .Salvianolic acid B is the root and rhizome of Salvia miltiorrhiza Bge, a traditional Chinese herb of HuoXueHuaYu. It can maintain the self-renewal of NSCs/ progenitor cells and promote the proliferation of NSCs though the regulation of the PI3K/Akt signaling pathway . It alsoin vitro [Tamethylpyrazine extracted from Ligusticum wallichii Franch can promote the proliferation and differentiation of NSCs under the condition of hypoxia in vitro , and alsin vitro .Panax notoginseng saponins (PNS), which has the effect of Huoxuehuayu, simultaneously promotes the expression of nestin/BrdU, improves the expression of tubulin-1 (tuj1), vimentin, and Nestin mRNA, and promote the self-renewal, proliferation, and differentiation of NSCs .Bilobalide extracted from Ginkgo biloba can increase the phosphorylation of the Cyclic AMP response element binding protein (CREB) in NSCs and the level of the neurotrophic factor, and promote the proliferation of NSCs .Berberine is the main active ingredient of Coptis chinensis Franch, a traditional Chinese herb of Qingrejiedu. Studies show that it can inhibit cell cycle arrest and promote the survival and differentiation of NSCs . Baicalein vitro and inhibit the apoptosis of cells. The mechanism may be related to down-regulation of the expression of inhibitor Κb (iκB), nuclear transcription factor-κB (NF-κB), and interleukin-1β (IL-1β)[Paeoniflorin is a natural compound extracted from the roots of the Chinese herbs Paeonia lactiflora Pall and Paeonia suffruticosa. It can promote the proliferation and survival of NSCs and precursor cells β (IL-1β).As mentioned above, the natural compounds extracted from traditional Chinese medicine, which play a regulatory role in the proliferation and differentiation of NSCs, can be divided into the three categories of tonifying , Huoxuehuayu, and Qingrejiedu herbs. The effects of tonifying herbs on NSCs proliferation and differentiation have been studied most. In the theory of TCM, the nerve function damage is often due to kidney deficiency caused by the deficiency of the marrow-reservoir. These tonifying herbs are therefore often used for the treatment of nervous system diseases. In addition, kidney essence is also believed to be critical in maintaining a variety of life activities. The potential function of various organs can be stimulated through the method of tonifying the kidneys, which is interlinked with the method of promoting the proliferation and differentiation of NSCs to repair brain neurons in the treatment of brain damage and neurodegenerative diseases. In addition, the insufficient cerebral blood supply caused by cerebral arteriosclerosis or atherosclerotic plaque inflammatory response is also a common cause of nervous system diseases. Therefore, the heat detoxification practice of TCM is also often applied to treat nervous system diseases, including the clinical use of Huoxuehuayu and Qingrejiedu herbs. Experimental studies also demonstrate that Chinese herbs and their active ingredients can regulate the proliferation and differentiation of NSCs, which provides a broader space for discovering drugs that can regulate the proliferation and differentiation of NSCs.NSCs pass through the different stages of NSPCs, NPCs, and neuroblasts in the process of proliferation and differentiation into neural cell lineage, and their corresponding markers are found at different stages. In the process of neurogenesis, and in NSC proliferation and differentiation, a variety of internal and external factors precisely regulate NSCs through different protein pathways. Studies have found that single herbs, herb extracts/Chinese herbal monomers, and compounds of Chinese medicine have a certain regulatory role on the proliferation and differentiation of NSCs. However, most of the current studies focus on single pathways. However, the regulation of the NSC proliferation and differentiation is involved in a complex signal network. In addition, NSC research into TCM lacks multi-targeted and multi-channel approaches, which is a systematic deficiency and thus they cannot fully explain the detailed mechanisms underlying regulating NSC by TCM. The effects of TCM promoting neurogenesis are still in the experimental stage, and may not be ready for the clinical application. In short, TCM should be played to its advantages, and in combination with modern medicine used to explore its potential in the regulation of neurogenesis to provide new possibilities for the treatment of brain damage and neurodegenerative diseases.


P = 0.003). Patients exposed to cisplatin generally showed PD-L1 up-regulation  compared to those not exposed to cisplatin . To validate these findings in vitro, changes in PD-L1 expression in HNSCC cell lines  were analyzed by western blotting and flow cytometry after treatment with cisplatin and interferon-gamma. In HNSCC cell lines, PD-L1 expression was significantly up-regulated after cisplatin, along with phosphor-MAPK/ERK kinase up-regulation. In conclusion, PD-L1 expression in HNSCC may be altered during cisplatin treatment, activating the MAPK/ERK kinase pathway.Programmed death-ligand 1 (PD-L1) expression is regarded as a predictive marker for anti-PD-1/PD-L1 therapy. The purpose of study was to explore the changes in PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) during treatment. Paired HNSCC tissues prior to and after cisplatin-based treatment were evaluated to determine PD-L1 protein expression by immunohistochemistry. Among the 35 HNSCC patient samples, PD-L1 expression status changed after treatment in 37.1% (13/35) of samples. Among the 13 patients whose baseline PD-L1 was negative, PD-L1 expression was increased in 9 cases (69.2%) and remained negative in 4 cases (30.8%,  Programmed death-ligand 1 (PD-L1) expression in tumors was recently introduced as a novel immunologic therapy for cancer . Cancer in vitro evidence has shown that the expression of PD-L1 in tumors changes in response to various exogenous signals including interferon-gamma, radiotherapy, or chemotherapeutic agents [The clinical use of PD-L1 as a predictive marker for PD-L1/PD-1 blocking agents is controversial for several reasons. The most important reason is that the expression of PD-L1 differs during the clinical course and tumor evolution process. Increasing c agents –12. PD-Lc agents , 14. Intc agents . TherefoWe hypothesized that PD-L1 expression changes during treatment in HNSCC patients. Thus, the aim of this study was to determine changes in PD-L1 expression during cisplatin chemotherapy in HNSCC cancer patients.Of the 35 HNSCC patients included in this study, baseline tumor tissue of 22 patients (62.9%) had PD-L1-positive tumors, while the other 13 patients (37.1%) had PD-L1-negative tumors (Table P = 0.003). In contrast, 4 of 22 patients (18.2%) who were initially PD-L1-positive tumor showed decreased PD-L1 expression  showed up-regulated PD-L1 expression after treatment (% showed P = 0.006) and vimentin expression was generally increased (P = 0.393) in post-treatment tumor tissue compared to in matched baseline tissues  and cisplatin-treated patients (83.3%). Previous clinical trials consistently showed that PD-L1 expression is an important biomarker for predicting anti-PD-1/PD-L1 treatment in various cancer types including HNSCC , 22; thuin vitro analysis to clearly define the association between the MEK pathway and PD-L1 up-regulation is needed.There were some limitations to the current study. First, only a small number of patients was analyzed. Moreover, although nearly a half of the patients received cisplatin chemotherapy, the sequence of treatment modalities and definitive treatment were very heterogeneous. Moreover, radiotherapy would also play a role in PD-L1 up-regulation in CCRT-treated patients. However, the main finding of the study was that PD-L1 changes according to treatment, which is important because it is difficult to obtain paired biopsy samples because of technical and ethical issues. In-depth In conclusion, PD-L1 expression in HNSCC is altered during the treatment phase, particularly following cisplatin-containing chemotherapy in baseline PD-L1-negative patients. A recent re-biopsy sample is required for precise evaluation of the PD-L1 expression status in HNSCC and to determine whether anti-PD-1/PD-L1 inhibitors should be used.Medical records were retrospectively reviewed to identify patients diagnosed with HNSCC who were treated at Seoul National University Hospital from December 2004 to November 2012. Patients from whom paraffin-embedded tumor samples were obtained prior to and after cisplatin chemotherapy were included.The decision to treat patients was determined by a multidisciplinary team , 24. Pat® mAb . IHC was performed using the Ventana Benchmark XT system . p16 was considered as positive when IHC analysis revealed diffuse and strong nuclear and cytoplasmic staining in ≥70% of tumor cells [Representative formalin-fixed paraffin-embedded tissue blocks from each case were subjected to immunohistochemistry (IHC) using the following antibodies: mouse anti-p16 (E6H4) monoclonal antibody (mAb) , mouse anti–E-cadherin (36B5) mAb , mouse anti-vimentin (V9) mAb , and rabbit anti-PD-L1 (E1L3N) XPor cells . PD-L1 Ior cells , 26. Cas2 at 37°C). Cells were resuspended in lysis buffer , incubated on ice for 10 min, and centrifuged for 15 min at 4°C. Samples containing equal quantities of total protein were resolved on SDS–polyacrylamide denaturing gels, transferred to polyvinylidene fluoride membranes, and probed with antibodies according to the manufacturer's protocols. Antibodies against PD-L1, p-MEK Ser217/221, MEK, p-STAT3, STAT3, E-cadherin, vimentin, and β-actin were purchased from Cell Signaling Technology. β-Actin was used as the protein loading control Detection was performed using an enhanced Lumi-Light Western Blotting Substrate kit .Head and neck cancer cell lines were purchased from the American Type Culture Collection  and Korean Cell Line Bank  and cultured as previously described . Briefly5 cells was aliquoted and placed into assay tubes. Next, 2 mL of fluorescence-activated cell sorting (FACS) buffer was added to each tube and rinsed twice by centrifugation. The cells were resuspended in 100 mL of FACS buffer with fixable viability dye . The cells were stained with PD-L1 phycoerythrin  or isotype control for 30 min on ice in staining buffer . Analysis was conducted using a FACSCalibur instrument  with CELLQuest software (BD Biosciences). Flow cytometric analysis of PD-L1 expression in head and neck cancer cells was conducted. After 24 h, the cells were harvested and stained with either mouse anti-human PD-L1 (clone 5H1) or a mouse IgG1 isotype control followed by PE-conjugated goat anti-mouse Ig.Flow cytometry was performed as previously described . A totalP values were two-sided and considered significant if P < 0.05. All statistical analyses were carried out using R version 3.1.2 (http://www.r-project.org).The chi-square test was used to determine the nature of the associations between PD-L1 positivity and clinicopathologic parameters. Overall survival was measured from the diagnosis date until death or the last follow-up date, if censored. Survival analyses were carried out according to the Kaplan-Meier method with log-rank testing to assess differences between groups. All reported The study protocol was approved by the Institutional Review Board of Seoul National University Hospital  and was conducted in accordance with the Principles of the Declaration of Helsinki.


In 2012, there were 22,200 residential care communities serving 713,300 residents across the United States. Forty percent of residential care communities were smaller with 4–10 beds, but these communities housed only 7% of all residents. The largest residential care communities with more than 100 beds were only 9% of all communities but housed 33% of all residents.Source: Caffrey C, Harris-Kojetin L, Rome V, Sengupta M. Operating characteristics of residential care communities, by community bed size: United States, 2012. NCHS data brief, no 170. Hyattsville, MD: National Center for Health Statistics; 2014. Available at http://www.cdc.gov/nchs/data/databriefs/db170.htm.Reported by: Vincent Rome, MPH, vrome@cdc.gov, 301-458-4466; Christine Caffrey, PhD; Lauren Harris-Kojetin, PhD.