The incorporation of pseudoallylic strain by N-methylation at the solvent exposed loop in proteins leads to a stark increase in their thermodynamic stability that can be tuned by altering the amino acid composition. N-methylation of the i+1–i+2 amide bond at the reverse turn results in well-folded and stable β-sheets in aqueous solution at room temperature. The developed superactive turn inducing motif is tolerant to a wide variety of functional groups present on coded amino acids making the designed turn fully compatible with bioactive loops in proteins. We demonstrate that the steric restraint and the functional groups at the reverse turn act in synergy to modulate the folding of re-engineered β-sheets. Introduction of the turn motifs onto a three-stranded β-sheet protein, Pin 1 WW domain, resulted in various analogs showing a cooperative two-state transition with thermal stability (TM) ranging from 62 °C to 82 °C. Despite modulating the stability of Pin 1 variants by ∼2.8 kcal mol–1 (ΔΔGf), the native fold in all the protein variants was found to be unperturbed. This structural stability is brought about by conformational preorganization at the engineered reverse turn that results in strong intramolecular hydrogen bonds along the three dimensional structure of the protein. Thus, this simple loop engineering strategy via two amino acid substitution provides us a “toolkit” to modulate the stability of β-sheet containing peptides and proteins in aqueous solution that will greatly expand the scope of de novo protein and foldamer design.Reverse turns are solvent-exposed motifs in proteins that are crucial in nucleating β-sheets and drive the protein folding. The solvent-exposed nature makes reverse turns more amenable to chemical modifications than α-helices or β-sheets towards modulating the stability of re-engineered proteins. Here, we utilize van der Waals repulsive forces in tuning the steric restraint at the reverse turn. The steric restraint induced upon  Tight packing in the hydrophobic core of a protein by optimizing the van der Waals interaction R-nipecotic acid-S-nipecotic acid,via synthetic and semi-synthetic methods.Free peptide bond rotation is a major driving force for the unfolding of a protein.i+1–i+2 amide bond at the turn by N-methylation.N-methylation was found to introduce a favorable pseudoallylic strain between various atoms of the i+1 and i+2 residues in the β-turn (CNMe/i–1)(Cβ and i)(CNMe/i) strictly favors a trans peptide conformation, whereas when present between two homochiral residues (l-/l- or d-/d-), there is an equal probability for the N-methylated peptide bond to adopt a cis or a trans conformation , the introduction of pA1,3 strain increases the Ktrans/cis in the heterochiral system (L3) and significantly decreases the Ktrans/cis in the homochiral system (L2). The reduced Ktrans/cis in the homochiral system is a result of the unfavorable 1,3-diaxial strain in the trans conformation, which is absent in the heterochiral system  alone does not provide sufficient conformational restriction to favor the trans conformation; however, in combination with the pA1,2 strain (L3) it increases the Ktrans/cis. These results clearly indicate that the differential behavior of an N-methylated peptide bond depends on the neighboring substituents with the highest propensity to adopt a trans conformation when the flanking residues are heterochiral.This finding encouraged us to evaluate the role of pseudoallylic strain in governing the peptides  using NMystem ESI. FurtherN-methylation of the peptide bond, we analyzed the Ramachandran plot of N-methylated linear tetrapeptides with varying pseudoallylic strain. We observed that irrespective of the chirality of N-methylated residue, N-methylation restrains Ψ of the preceding residue , due to van der Waals repulsion, provides added stability to the β-sheet.Initially, we synthesized four molecules with varying pseudoallylic strain in the reverse turn to analyze the critical role of tability . We acquand 2, 3 , we obse > 4 > 1  which isments ESI. This ordom coil .20 We al peptide .21 The bi+1 and i+2 residues of the reverse turn in the β-sheet peptide with diverse functional groups, but fixed N-methylation at the i+1–i+2 amide bond.Encouraged by this finding, we set out to systematically scan the 3 that showed the maximum foldedness. Since the pseudoallylic strain pA1,3 is critical in folding the peptide, keeping the i+1 substitution constant, we initially sought to substitute the i+2 residue with various l-amino acid side chains and assess their influence on the β-sheet stability using ∑CSD  between i+2 N-methyl and i+2 Cγ  leads to destabilization of 3b and 3a. In contrast, in 3e the β-branch demonstrates almost a two-fold increase in the ∑CSD resulting from the restricted χ1 rotation due to a probable intramolecular hydrogen bond between Thr–OH and its C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbdri0Nd" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O , we obtained a more than two-fold increase in the ∑CSD compared to that of 3b. Another notable observation was the increase in the foldedness of the β-sheet by incorporating positive charges, e.g., in 3l and 3n at the termini of an unbranched amino acid side chain 3f. These results show how modulation of local steric interactions and charges at a single site in the β-turn can alter the overall foldedness of the β-sheet.Our primary library was based on ing ∑CSD .21 The cing ∑CSD  indicateg>O ESI. Moreovei+2 substituted linear peptides, we estimated the folded population of selected β-sheets and their free energy of folding (ΔGfold) in water at 25 °C . The presence of a hydrophobic β-branch (3a and 3b) is detrimental to the folding of the peptide, whereas a –CH3 to –OH substitution dramatically alters the folded population . Thus, we observe that a combination of pA1,2 and polar functional groups enhances the stability of the β-sheet by –1.0 kcal mol–1 (ΔΔGf between 3n and 2).Next, towards the thermodynamic analysis of at 25 °C  utilizind-amino acid substitutions at the i+1 site with N-methylated alanine at the i+2 site. To our satisfaction, we observed the strong tolerance of various functional groups at the reverse turn (i+2 site (3b), by virtue of the unfavorable pA1,2 strain destabilizes the β-sheet, whereas when present at the i+1 site, the favorable pA1,3 strain enhances the stability of the β-sheet (4g) which is comparable to the stability of the ring constrained reverse turn motif pG (–1 destabilization (ΔΔGf between 3 and 3b) or stabilization (ΔΔGf between 3 and 4g) when present at the i+2 or i+1 site, respectively . To our amazement, the introduction of the i+1 isopropyl group into 3n resulted in an additional 0.58 kcal mol–1 stabilization (ΔΔGf between 5 and 3n) yielding a super stable monomeric β-sheet 5. This result suggests that the i+1 and i+2 substituents act synergistically to enhance the foldedness of the engineered β-sheet. Additionally, although almost all functional groups are tolerated at the reverse turn, certain amino acids, leucine, lysine, glutamine, and arginine at the i+2 site and d-aspartate, d-threonine, d-lysine, d-tyrosine, d-phenylalanine and d-valine at the i+1 site, display high propensity to stabilize the β-sheet  < –2.3 ppb K–1 (2) < –1.6 ppb K–1 (3) < –1.0 ppb K–1 (3n) ≅ –1.0 ppb K–1 (4g) < –0.6 ppb K–1 (5)). Thus, it is tempting to speculate that the increased conformational restriction in the β-turn possibly results in an enhanced strength of the intramolecular hydrogen bond stabilizing the β-turn.Thus, this conformational restriction results in a well-formed β-turn with a strong hydrogen bond between Lys8Hin.Gfold  corrobor3n and 5 typical of β-sheet rich proteinsi+2 site in the reverse turn. The observed right-handed twist was also indicated by the presence of strong NOEs between Lys9Hβ/γ and Tyr2Hδ in the ROESY spectrum3n and 5 as opposed to 4g , also shows the involvement of the guanidinium group of arginine at the i+2 site in side chain-backbone hydrogen bonding.Another remarkable feature was the stark right-handed twist observed in to 4g ESI.28 Althoi+1 and i+2 side-chain functional groups which imposes a favorable steric constraint on the β-turn. Such preorganization of the main chain torsion angles by side-chain substitution confers extraordinary conformational stability to proteins and peptides as was observed in collagen triple helices by substituting proline with 4-fluoroproline and 4-methylproline.32The enhanced thermodynamic stability of the monomeric β-hairpins in water is fundamentally attributed to the conformational preorganization of the β-turn attained by different et al. that the introduction of a type II′ β-turn affords the fastest folding of the WW domain.This encouraged us to utilize the designed turn motifs with myriad side-chain functional groups to engineer the conformational stability of proteins. Since antiparallel β-sheets are quite prevalent in proteins and have found great utility towards the development of non-antibody based- and hyperstable peptide scaffolds,TM and free energy of folding (ΔGf), respectively. Unlike the model 12-mer peptide, the incorporation of pA1,3 strain at the reverse turn in 6 was sufficient to impart reasonable thermodynamic stability (TM of 68.7 ± 0.2 °C) to the Pin 1 protein due to multiple hydrophobic interactions along the β-stands in the entire protein. The TM values of all the Pin 1 variants studied were independent of the protein concentration over a range of 5–50 μM indicating their monomeric nature  in 7 (i+2 site (8) by virtue of the unfavorable pA1,2 strain destabilizes the Pin 1 by ∼0.9 kcal mol–1 (ΔΔGf). However, when incorporated at the i+1 site (9), it stabilizes the protein by ∼1.2 kcal mol–1 (ΔΔGf), further strengthening the critical role of steric interaction at the reverse turn in governing the thermodynamic stability of proteins. Nevertheless, the most exciting observation was the synergism between i+1 and i+2 residues at the reverse turn in enhancing the thermodynamic stability of the Pin 1 protein as observed in 10  and 11  with the incorporation of lysine and arginine at the i+2 site, respectively, which corroborated our findings in the 12-mer model peptide.All the Pin 1 variants were chemically synthesized, which showed the characteristic maximum at 227 nm in CD spectra indicating the presence of a folded protein ESI. Their tature ESI. FurtherGf) in 7 . The int8 and 11 (ΔTM of ∼20 °C) corresponding to about 2.8 kcal mol–1 (ΔΔGf) results from significant perturbation in the secondary structure of the re-engineered Pin 1. To identify the structural alterations in the solution conformation of Pin 1 variants, we acquired the 1H, TOCSY, and NOESY spectra of each Pin 1 variant (40–60 μM) at 15 °C. On comparing the backbone Hα CSD of the re-engineered Pin 1 variants with the wild-type apo-Pin 1 WW domainNG)1H resonances  were observed in Arg11 show strong hydrogen bonding as compared to 8 to the amino acids of the flanking N-term and C-term strands indicating higher stability of the intramolecularly hydrogen bonded region in 11.Overall, we observed more positive amide temperature coefficient values in 1 than 8 ; howeverN-methylation of a peptide bond differentially modulates the population of cis/trans isomers about the tertiary amide bond. This property of an N-methylated peptide bond along with its heavily restricted conformational space allowed us to probe the strength of pseudoallylic strain pA1,2 and pA1,3 in engineering reverse turns with different functional groups present on coded amino acids. Utilizing the combination of pseudoallylic strain and the functional groups on coded amino acids, we could impart remarkable conformational stability to isolated β-sheets and proteins in aqueous solution at room temperature. This enhanced conformational stability is presumably achieved by preorganization of the β-turn by the formation of stable hydrogen bonds at the reverse turn in the transition state.i+1 and i+2 sites of a β-turn, which induces favorable steric constraints upon the main chain. Thus, by modifying the side-chain substituents at the turn, we could successfully tune the stability of β-sheets. Interestingly, a single amino acid substitution at the reverse turn also contributes towards modulating the right-handed twist in isolated β-sheets.In summary, we demonstrate that the non-covalent constraint induced by Gf of ∼2.8 kcal mol–1 between the least and the most stable Pin 1 analogs). Hence, with the remarkable advancement in tools to generate semi-synthetic proteins45Since reverse turns are solvent exposed, and the pseudoallylic strain provided a basic conformational stability to the reverse turn, it allowed us to probe the effect of various amino acid substitution on the stability of a mini-protein, Pin 1 WW domain. We show that re-engineering the non-repetitive motif like β-turns with non-natural amino acids can have a large contribution to protein stability (ΔΔThere are no conflicts of interest to declare.Supplementary informationClick here for additional data file.


The transport properties of few-layer graphene are the directly result of a peculiar band structure near the Dirac point. Here, for epitaxial graphene grown on SiC, we determine the effect of charge transfer from the SiC substrate on the local density of states (LDOS) of trilayer graphene using scaning tunneling microscopy/spectroscopy and angle resolved photoemission spectroscopy (ARPES). Different spectra are observed and are attributed to the existence of two stable polytypes of trilayer: Bernal (ABA) and rhomboedreal (ABC) staking. Their electronic properties strongly depend on the charge transfer from the substrate. We show that the LDOS of ABC stacking shows an additional peak located above the Dirac point in comparison with the LDOS of ABA stacking. The observed LDOS features, reflecting the underlying symmetry of the two polytypes, were reproduced by explicit calculations within density functional theory (DFT) including the charge transfer from the substrate. These findings demonstrate the pronounced effect of stacking order and charge transfer on the electronic structure of trilayer or few layer graphene. Our approach represents a significant step toward understand the electronic properties of graphene layer under electrical field. The peculiar band structure features of massive Dirac fermions in multilayer graphene are driving intense activity in fundamental research, as well as for applications in next generation optoelectronic devicesπ and π* bands near the Fermi level in monolayer graphene are modified in FLG, showing strong dependence on the stacking sequence as well as the number of layer4P6/mmm), ABA stacking , and ABC stacking 8In few-layer graphene (FLG), the interlayer coupling introduces perturbations of the low-energy band dispersions. Consequently, the linear Among the different methods to obtain few layer graphene, SiC graphitization is a viable method for a large-scale graphene production11Here, large-area uniform trilayer graphene layers were successfully synthesized on off-axis 4H-SiC(0001). The morphology of the trilayer graphene consisting of large and flat domain was confirmed by LEEM. The band structure and the charge transfer from the substrate to graphene layer were studied using ARPES. The electronic properties and the stacking order trilayer graphene are studied by STM/STS and density functional theory (DFT). Our studies indicate that trilayer epitaxial graphene exhibits two structure with Bernal  and rhom−1. The peak at 1522 cm−1 is considered to be an overtone of the L-point optical phonon from the SiC. The Raman signals from the graphene show prominent characteristic peaks at 1595 cm−1 (G) and 2705 cm−1 (2D), which gives evidence of carbon sp2 reorganization. The G peak value of 1595 cm−1 indicates n type doping. Also the blue-shifted position of the 2D peak at 2705 cm−1 could be the result of a compressive strain of the graphene layer during the post-growth cooling down procedure or charge transfer doping from the substratefwhm) of about 75 cm−1. This broadening can be attributed both to a defect scattering and to the formation of two or more graphene layers. The intensity ratio of the defect-induced D band to the graphite G band (ID/IG) has been widely used to estimate the density of defects in grapheneLa)laser is the Raman excitation wavelength (λlaser = 532nm) and the measured ratio μm. The regions with two different graphene thickness can be distinguished by differences in the reflected intensity25The trilayer graphene used in this study was obtained by annealing 4° off-axis 4H-SiC(0001) at 1550 °C in 800 mbar argon for 10 min. We used micro-Raman spectroscopy to probe the quality and electronic properties of the sample. p spectrum. The spectrum was fitted by sums of Voigt curves . The Lorentzian width (fwhm) was fixed at 45 meVp peaks are made up of spin-orbit split doublets  substrate while the component at 100.9 eV is attributed to the presence of Si clusters formed when Si-C bonds are broken during graphitization301s spectrum showed three components at 283.4, 284.5, and 285.3 eV in binding energy. The sharp C 1s peak, labeled G, located at 284.5 eV in binding energy, indicates the presence of sp2 hybridized C–C bonds and is the signature of the graphene layers. Due to its metallic nature this component was fitted using a Doniach-Sunjic line shape with an asymmetry factor α of 0.1 and a fwhm of 0.5 eV. The two other peaks labelled SiC (283.4 eV) and IL (285.3 eV) are attributed to the SiC bulk and to the interfacial layer respectively. Due to the non-metallic nature of these components, the peaks are symmetric (asymmetric factor α = 0 eV). From the C 1 s spectrum, the thickness of the graphene layers can be calculated from the ratio between the intensities of the G and SiC components. This ratio corresponded to an exponential decay of roughly 3 ML of carbon coverage supposing a continuous and homogenous coverage of the surfaceThe chemical bonding environment of the sample has been probed using X-ray Photoelectron Spectroscopy (XPS) experiments. High-resolution spectra of Si 2p and C 1 s recorded under surface sensitive conditions  are shown in F and ED  surface. The surface is formed by self-ordered periodic structures consisting of pairs of a (0001) basal plane terrace and a (1–10n) nanofacet with a characteristic periodicity of ∼600 nm, as shown in  zone A,  of Fig.  zone B, . The dif zone B, . From th−3 electrons per unit cellD in the 150–200 meV range. z orbitals of the uppermost graphene layer. That this is a reasonable approximation follows from the fact that the STM signal is proportional to the LDOS integrated between the Fermi energy and the corresponding bias voltagez orbitals and since the pz orbitals of the uppermost layer of the multilayer sample have the largest extend into the vacuum region they will contribute most to the signal. We also verified this for the ABC trilayer by calculating the numerical derivative of the STM signal as obtained with the Tersoff-Hamann approximationdI/dV versus bias (V) spectra and the calculated LDOS shown in e.g in the case of ABCA stacking, the variation in charge transfer induces different intensity ratio between the peak above the Dirac point and the secondary peaks at 200–300 meV.In order to understand how the tunneling spectra depend on the stacking of the layer we compare the STS results with the local density of states (LDOS) as calculated by DFT. This comparison reported in α or β atoms of the outermost layers. The DFT studies taking into account the doping of graphene due to the interaction with the SiC underlayer are able to reproduce the observed STS results which are different from the case of undoped rhombohedral stacked graphene. The present analysis is only possible because the STM/STS are very sensitive to the stacking, and because of the very high energy of the STM/STS experiment. Note that the total energy difference between ABA and ABC stacked trilayer graphene is very small (0.18 meV/atom)As compared to the Bernal stacking, the rhombohedral stacked few layer graphene exhibits band dispersions near the Dirac point. Specifically, the low-energy bands near the Dirac level (flat band) in ABC-stacked few layers graphene has a surface states with its wave functions distributed on either In summary, we identify the three bands due to interlayer interactions and two stacking sequences of the trilayer graphene using ARPES and STM/STS measurements. We find that the stacking and the charge transfer change significantly the LDOS of the few layer graphene, which illustrate the unique electronic properties of graphene layers. The STS and DFT spectra show a clear evidence for flat bands near Dirac energy in trilayer graphene on SiC. The flat bands, which contribute to a pronounced peak in the tunneling density of states, arise from ABC stacking. The morphology of the trilayer graphene consisting of large and flat domain was confirmed by LEEM. The lateral domain size is only limited by the step edges pre-existing in the substrate. STS showed the presence of epitaxial trilayer graphene with a two- stacking domain arrangement of carbon atoms.n-type 4H-SiC(0001) substrates that were etched in hydrogen at 1550 °C and annealed at 1525 °C in argon partial pressure of P = 800 mbar. The samples were then cooled down to room temperature and transferred ex-situ from the growth chamber to undergo STM/STS, XPS/ARPES measurementsThe few layers graphene were synthesized on off axis T = 4.2 K, the I(V) characteristics were acquired while the feedback loop was inactive, the differential conductivity dI/dV , proportional to the LDOS, was measured directly by using a lock-in technique. For this purpose a small AC modulation voltage Vmod was added to V  and the signal dI detected by the lock-in amplifier was used to determine the differential conductivity dI /dVmod.STM/STS measurements were carried out using an Omicron ultra-high vacuum low temperature scanning tunnelling microscope (UHV-LT-STM)XPS/ARPES measurements were conducted at the TEMPO beamline of the SOLEIL synchrotron 2 electron-momentum mesh with an 1 mRy Methfessel-Paxton first-order spreading2 electron-momentum grid. In all figures of the paper, the DFT band energy was rescaled by a 1.18 multiplicative factor to correct for the DFT underestimation of the Fermi velocityThe electronic structure calculations were performed using the QUANTUM-ESPRESSO codeHow to cite this article: Pierucci, D. et al. Atomic and electronic structure of trilayer graphene/SiC(0001): Evidence of Strong Dependence on Stacking Sequence and charge transfer. Sci. Rep.6, 33487; doi: 10.1038/srep33487 (2016).


Particle-attached (PA) and free-living (FL) microorganisms play significant but different roles in mineralization of organic matter (OM) in the ocean. Currently, little is known about PA and FL microbial communities in bathyal and abyssal pelagic waters, and understanding of their diversity and distribution in the water column and their interactions with environmental factors in the trench area is limited. We investigated for the first time the variations of abundance and diversities of the PA and FL bacterial communities in the epi-, bathy-, and abyssopelagic zones of the New Britain Trench (NBT). The PA communities showed decreasing species richness but increasing relative abundance with depth, suggesting the increasing ecological significance of the PA bacteria in the deep ocean. The abundance and diversity of PA and FL bacterial communities in the NBT water column appeared to be shaped by different sets of environment factors, which might be related to different micro-niches of the two communities. Analysis on species distribution suggested that the differences between PA and FL bacteria communities mainly resulted from the different relative abundance of the “shared taxa” in the two types of communities. These findings provide valuable information for understanding the relative ecological roles of the PA and FL bacterial communities and their interactions with environmental factors in different pelagic zones along the vertical profile of the NBT water column. The pelagic deep ocean contains 70% of the ocean’s microbial cells and 60% of its heterotrophic activity, playing a significant role in regulating the biogeochemical cycles of the earth . About 1The physical and chemical conditions in the water column of the ocean are not uniform, and great variations exist for environmental factors with increasing depth . These fThe hadal trenches are the deepest part of the ocean. With unique tectonic, topographic, bathymetric and hydrographic features, trenches have been recently discovered as hotspots of microbial activities in the deep ocean , probablIn this study, we investigate for the first time the changes of abundance and diversity of the PA and FL bacterial communities in the water column of the New Britain Trench (NBT). Water samples were collected along the vertical profile from the surface to the abyssopelagic zone. The objectives are to (1) determine the abundance and diversity of PA and FL bacterial communities from the surface to the abyssopelagic zone; (2) reveal the succession of PA and FL bacterial taxa in response to environmental factors in the water column and the implications for biogeochemical cycles in the oceans.Zhang Jian. The full depth of this station is 6700 m, and water samples were collected from 75, 200, 1000, 2000, 3000, 4000, 5000, and 6000 m using Niskin bottles fitted on a Sea-Bird Carousel equipped with a conductivity-temperature-depth (CTD) sensor (Sea-Bird SBE 911plus). For each depth, around 50 L of water was taken and was processed immediately onboard the ship: (1) duplicate 20 L of the water were filtered sequentially through 3 and 0.2 μm polycarbonate filters  to collect PA and FL microbes, respectively. Duplicated filters were obtained for each size fraction and were immediately stored at -80°C; (2) for POC and PON analysis, 7–9 L of the water was filtered through 0.7 μm nominal pore size glass fiber filters  which were pre-combusted in a muffle furnace at 500°C for 12 h. The membrane filters were stored at -20°C after filtration. The filtrates were also stored at -20°C for DOC analysis; (4) Triplicated 100 mL of the water were fixed in paraformaldelhyde  and stored for later microscopic counting of the microbial cells; (5) The remaining water was stored at -20°C for inorganic nutrient measurements.Water samples were taken in August 2016 at station W  in the western New Britain Trench during a cruise aboard M/V 3-, NO2-, NH4+, and PO43-) were determined using a QuAAtro autoanalyzer .Physical characteristics of the water including depth, salinity and temperature were measured with CTD sensors during sampling. Concentrations of POC and PON were determined with a PE2400 Series II CHNS/O analyzer  . DissolvIn laboratory, PFA fixed cells were filtered onto 0.22 μm pore size black polycarbonate filters  and stained with DAPI . Bacterial cells were counted under epifluorescent microscopy . For each sample, no less than 20 microscopic fields were counted and cell abundance was calculated according to In this study, we applied two different DNA extraction methods and two primer sets targeting different hypervariable regions of 16s rRNA gene. Two sets of data were generated for each of the samples, and were then combined into one data set according to the method described in a later section about sequences analysis.-1) and then extracted with AllPrep DNA/RNA Mini Kit (Qiagen). The 2nd filter was extracted with FastDNATM SPIN Kit for Soil DNA Extraction (MP) according to manufacturer’s protocols. The concentration and quality of final DNAs were determined by NanoDrop 2000 UV-vis spectrophotometer . DNA samples from the 1st replicates were amplified with barcoded primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) targeting the V3–V4 hypervariable regions. DNAs from the 2nd replicates were amplified with barcoded primers 515F (5′-GTGCCAGCMGCCGCGG-3′) and 907R (5′-CCGTCAATTCMTTTRAGTTT-3′) targeting the V4–V5 hypervariable regions of 16s rRNA gene. PCR reactions were conducted using a thermocycler PCR system  with the following program: 5 min of denaturation at 95°C, 27 cycles of 60 s at 95°C, 60 s at 55°C for annealing, and 60 s at 72°C for extension, and a final extension step at 72°C for 10 min. PCR reactions were performed in triplicate 50 μL mixture with each containing 1X ExTaq buffer, 0.2 mM of each dNTP, 0.2 μM of each primer, 1 U of ExTaq polymerase  and 1 μl DNA. PCR products were purified from 2% agarose gel using AxyPrep DNA Gel Extraction Kit  and quantified using QuantiFluorTM-ST . DNAs from PA and FL fractions of 3000 m and PA fraction of 5000 m were failed in PCR amplification with 338F/806R, so these samples were excluded from the diversity analysis.For each depth, duplicate PC filters were obtained for both 3 and 0.2 μm fractions (as described above). The 1st filter was lysed using lysozyme (30 mg ml1. The taxonomy of each OTU was assigned by RDP Classifier2 against the Silva16S rRNA database (SSU123) with a confidence threshold of 70%.Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 300) on an Illumina MiSeq platform  according to standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. . Raw fastq files were demultiplexed, quality-filtered by Trimmomatic  and asseTwo datasets, named “Qiagen-338-806” and “MP-515-907” were generated for each of the samples, and were combined into one dataset according to The diversity indices including species richness (at OTUs level), Pielou’s evenness, Shannon index, Simpson index were calculated with PRIMER 6.0 package using OTU table of the merged data set generated as described above. Venn diagram was produced with VENNY 2.1  to show ® qPCR assay Eub338F/Eub518R (® qPCR Master Mix (Vazyme). Duplicate reactions were used for each sample. The thermal cycles on the ABI 7500 (Applied Biosystems) were the following: initial denaturation (95°C) for 5 min, followed by 40 cycles of denaturation (95°C) for 5 s, annealing at 55°C for 30 s and extension at 72°C for 40 s. A melting curve analysis was included in each run of qPCR to check the specificity of amplification. Cycle of threshold (Ct) was calculated using the ABI 7500 software with the auto-baseline and auto-threshold functions. Standard curves of Ct vs. gene copy numbers were constructed using serial dilutions of linearized plasmids containing amplicon fragment of Eub338F/Eub518R.Each DNA sample was tested with SYBR Green/Eub518R  to deterFigure 1A). Nitrate and phosphate concentrations were the lowest at surface water and the highest at around 1000 m, while the concentration of nitrite fluctuated along the whole water column with a decreasing trend . POC and PON concentrations were continuously decreasing with increasing depth. DOC showed similar trend, but its concentration remained relatively constant below 1000 m . Microbial cell abundance ranged from 3.14 ± 1.61 × 105 cells/ml in surface water to 7.17 ± 9.23 × 103 cells/ml at 2000 m and remained relatively constant from 2000 to 6000 m with an average value of 9.75 × 103 cells/ml . It is interesting to note that the relative abundances of PA and FL bacteria, as determined by qPCR quantification of bacterial 16s rRNA genes, showed reversed vertical trends. The relative abundance of PA bacteria increased and that of FL bacteria decreased with depth .Temperature continuously decreased from 28.4°C at surface water to around 2.2°C at 2000 m and maintained at around 1.9–2.3°C from 2000 to 6000 m. Salinity showed gradual increase from 34.4 PSU at surface to 35.7 PSU at 180 m, decreased to 34.5 PSU at 1000 m, and then maintained at around 34.5–34.7 PSU until 6000 m . Different from species richness, changes of Pielou’s evenness and Shannon and Simpson indexes showed similar patterns of vertical variation for both PA and FL bacterial communities, with lowest value at 1000 m and highest values at 200 and 6000 m . The relative abundance of Bacili class gradually increased with depth and was constantly higher in the PA than the FL communities in the whole water column . The relative abundances of Actinobacteria and Flavobacteria were consistently higher in the PA than in the FL bacterial communities in waters deeper than 200 m . In contrast, the SAR406 and SAR 202 clades displayed much higher relative abundances in the FL than in the PA communities in deep waters .Proteobacteria was the single dominant phylum in the entire water column of the NBT, accounting for 41.8–82.7% of the total sequences retrieved from different depths, with relatively higher proportions at 1,000 – 4,000 m depth and lower proportions at surface and abyssal depth . Overall, the samples were clustered into three large groups, roughly representing different pelagic zones along the depth profile, i.e., the epipelagic zone (75 and 200 m), the bathypelagic zone , and the abyssopelagic zone (5000 and 6000 m). It was also clear that, within each pelagic zone, the respective PA and FL bacterial communities tend to form different groups . Analysis of similarity (ANOSIM) supported the nMDS groupings: the differences between the pelagic zones were significant , while the differences between the PA and FL fractions in the whole water column were much lower , suggesting that vertical depth stratification is more important than trophic lifestyle in shaping the species composition of bacterial communities in the water column of the NBT.Non-metric multidimensional scaling (nMDS) and hierarchical clustering (UPGMA) analyses showed obvious differences in species composition between the communities . The results showed that the first two CCA axes explained 55.9% of the variations for species composition of the PA communities, and 66.1% of the variations for the FL communities. Monte Carlo test on significance of the first CCA axes showed that only the CCA ax for FL communities was statistically significant (P = 0.016) while that for the PA communities was not (P = 0.053). Among the tested environmental factors, phosphate was the statistically significant variable associated with the vertical variations of the PA bacterial communities (P = 0.031), while pressure (P = 0.008) and PON concentration (P = 0.009) were the statistically significant variables associated with the vertical pattern of FL bacterial communities.Canonical correspondence analysis was further conducted to show the correlations between the major environmental factors and the observed differences in species composition between the PA and FL bacterial communities . However, when taking into consideration of the relative abundance of each OTU in total sequence reads of different samples, we found that OTUs commonly present in both the PA and FL communities accounted for 61.0–95.3% of the total reads of the samples, with an average of 82.2% . In other words, on average 82.2% of the reads from the tested samples belonged to OTUs that present in both the PA and FL bacterial communities.Venn analysis showed that the proportion of the shared OTUs between the PA and FL bacterial communities ranged between 22.8% (at 1000 m) to 52.9% (at 200 m), with an average of 32.3%; while the proportion of OTUs present exclusively in the PA communities ranged between 17.4% at 6000 m and 49.1% at 75 m ; and that of OTUs present exclusively in FL communities ranged between 15.9% at 75 m and 54.6% at 6000 m, with an average of 34.9% . As a result, 17, 13, and 14 indicative OTUs were identified from the epipelagic, bathypelagic and abyssopelagic zones, respectively. The OTUs had uneven distributions in the PA and FL bacterial communities, but most of them presented in both types of the bacterial communities. For instance, among the identified indicative OTUs, 16 from the epipelagic zone, 11 from the bathypelagic zone, and 9 from the abyssopelagic zone were present in both the PA and FL bacterial communities; only minor proportions of indicative OTUs were present exclusively in either the PA or FL communities  than FL (0.0–3.0%) communities  and FL (6.3–32.8%) communities throughout the water column, and their preference of lifestyle varied at different depth .SIMPER analysis was applied to identify the “indicative OTUs” that were responsible for the observed dissimilarities between the PA and FL communities in the NBT water column. In this study, “indicative OTUs” were defined as OTUs that contributed more than 1% dissimilarities between the two bacterial communities , indicating the potentially increasing ecological significance of PA bacterial communities in the deep ocean comparing to shallower depths. This result could also partially explain the accumulating findings in the literature that bacterial communities in the deep ocean are typically enriched for taxa and genomic features with a particle- or surface-associated lifestyle . As a result, species richness was higher in PA than FL bacterial communities in the surface water (epipelagic zone), and higher in FL than PA communities at the bathyal and abyssal depths . Our results for the epipelagic water are consistent with previous studies conducted at similar depths . ANOSIM analysis showed that there were significant differences in microbial communities between the pelagic zones and no significant differences between communities of the two trophic lifestyles  in the whole water column (Table 1). These results suggest that vertical stratification is a key factor in shaping bacterial community structure in the NBT water column.Beta diversity analysis showed that bacterial communities in the NBT water column mainly clustered according to the pelagic zones. However, the PA and FL communities within each zone tend to grouped separately . This finding suggests that the FL bacterial communities are likely more responsive to the depth-related variations of environmental factors tested in this study. Consequently, the magnitude of changes in bacterial diversity with depth was much larger for the FL communities than that for the PA communities , further supporting the potential significance of pressure-adapted piezophilic bacteria and their role in mediating biogeochemical cycles in the deep ocean , but much less so for the PA bacterial communities (55.9%) . The majority of the sequences (61.0–95.3%) belonged to OTUs that were shared between the PA and FL communities . We further analyzed the major OTUs responsible for discriminating the PA and FL bacterial communities (the “indicative OTUs”). Our results suggest that the majority of the “indicative OTUs” are those that were shared between the PA and FL communities and that had much higher relative abundances in one type of the communities . In other words, the differences in community structure between the PA and FL bacterial communities tested in this study are mainly due to the uneven distribution of the shared OTUs in the PA and FL fractions. High percentage of sequences belonging to shared OTUs in PA and FL bacteria have also been observed in several previous studies in surface water . The enrichment of different bacterial taxa on PA or FL lifestyle was more obvious at the class level: the PA community contained higher proportions of species belonging to Cyanobacteria, Bacilli (Firmicutes), Flavobacteria (Bacteroidetes) and Actinobacteria throughout the water column . Due to the phototrophic nature of Cyanobacteria, these bacteria may not be playing an active role in the deep water, but the presence of Cyanobacteria in the PA bacterial communities throughout the water column may serve as an indication of bacteria attachment to and vertical transport of particles and the attached bacteria. It is also possible that this represents an artifact of filtration as the size of certain Cyanobacteria taxa is much greater than the pore size of the filter we utilized (3 μm). Alternatively, the detected Cyanobacteria might be dead cells and serve as substrates for PA bacteria, which are transported to the deep water. Association of taxa from Firmicutes and Bacteroidetes with PA lifestyle has been reported in the bathypelagic oceans globally . Enrichment of these two groups in the FL fraction of the deep ocean has also been reported in previous studies  under accession ID SRP125714.All raw sequences used in this study are publicly available at the NCBI Sequence Read Archive (SRA, RL, LW, and JF designed the expedition and sampling scheme, analyzed the data, and wrote the article. QL and RL prepared the samples on board. LW, QL, ZW, and ZL conducted the experimental procedures including cell counting, DNA extraction, PCR amplification, qPCR analysis, and bioinformatics analysis. ML measured all chemical parameters. RL, LW, QL, ZW, ZL, JF, and LZ made comments and suggestions to improve the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Structural immunology, focusing on structures of host immune related molecules, enables the immunologists to see what the molecules look like, and more importantly, how they work together. Antibody-based PD-1/PD-L1 blockade therapy has achieved brilliant successes in clinical applications. The recent breakthrough of the complex structures of checkpoint blockade antibodies with their counterparts, pembrolizumab with PD-1 and avelumab with PD-L1, have made it clear how these monoclonal antibodies compete the binding of PD-1/PD-L1 and function to blockade the receptor-ligand interaction. Herein, we summarize the structural findings of these two reports and look into the future for how this information would facilitate the development of more efficient PD-1/PD-L1 targeting antibodies, small molecule drugs, and other protein or non-protein inhibitors. Another representative example of structure-based drug design is the tyrosine kinase inhibitor, imatinib mesylate , to treat chronic myeloid leukemia as well as other blood neoplasias and solid tumors with etiologies based on activation of these tyrosine kinases.4 As an important subfield of structural biology, how will the structural immunology contribute to drug design to modulate immune responses is considered to be a key issue to promote translational medicine in the field.The late structural biologist and crystallographer Don C. Wiley once said, ‘I don’t believe the biology until I see it’.6 T-cell activation involves multiple paired molecular interactions including T-cell receptor (TCR)/peptide major histocompatibility complex (pMHC) interactions, CD4 (or CD8)/pMHC co-receptor interactions and co-stimulatory ligand-receptor interactions under the current two-signal system theory  is a member of the CD28 superfamily and was first discovered as a gene upregulated in a T-cell hybridoma undergoing cell death, therein the name was originated.13 PD-1 ligand 1 (PD-L1) and PD-1 ligand 2 (PD-L2) were identified to be the ligands (PD-Ls) of PD-1.15 Studies show that co-inhibitory molecules such as PD-1 and PD-L1 induce immune suppression in the tumor microenvironment which subsequently leads to the tumor immune escape  take advantages of specific binding to antigens with its complementarity-determining region (CDR) loops of both heavy chain (VH) and light chain (VL) and immune activating mediated by fragment crystallizable (Fc) region  region , and thu29 And is there any possibility that we could design a better therapeutic MAb targeting the hot spots and avoid antigen mutational escape? All these questions remain unanswered.Although MAb based immunotherapy has achieved great successes in fighting against multiple tumors in clinical application, some basic questions and concerns yet exist. It is unclear how the MAbs interact with PD-1 or PD-L1 to block the interaction of PD-1/PD-L1, though dominant negative competition for the receptor-ligand interaction is proposed. Are there any hot spots in PD-1 or PD-L1 for checkpoint blockade MAb targeting? Can we predict the possible mutational escapes on PD-1 or PD-L1 under the immune selective pressure of the MAbs during immune checkpoint blockade therapy in the future? Though mutational escape in PD-1 or PD-L1 has not been reported, CD19 mutational escapes or alternative splicing under chimeric antigenic redirected T-cell immunotherapy have already been observed.30–32 As a PD-L1 targeting antibody, avelumab is a human IgG1 antibody co-developed by Merck  and Pfizer, which is now in multiple phase III clinical trials against non-small cell lung cancer (NSLC) (NCT02395172), advanced renal cell cancer (NCT02684006) and gastric cancer (NCT02625610). Complex structure of avelumab/hPD-L1 shows that the avelumab utilizes both VH and VL to bind to the IgV domain of PD-L1 on its side  of pembrolizumab to hPD-1 and avelumab to hPD-L1 are 27.0 and 42.1 pm, respectively.31 On the other hand, the binding affinity between hPD-1 and hPD-L1 is 0.77–8.2 μm,33–35 which is much weaker than that of the MAbs. The strong binding of pembrolizumab to hPD-1 and avelumab to hPD-L1 would enable the binding priority of the therapeutic MAbs with checkpoint molecules and subsequent blockade of the hPD-1/hPD-L1 interaction.The recently reported avelumab/hPD-L1 and pembrolizumab/hPD-1 complex structures by our group and Song group have provided clear structural information on how the therapeutic MAbs abrogate the binding of PD-1/PD-L1.its side .30 The VVL chain . The mec of PD-1 . Though 36 Second, therapeutic MAbs maintained the Fc fragment would induce cytotoxic immune responses even engineered with IgG4 class or antibody-dependent cell-mediated cytotoxicity (ADCC) defective mutations.38 Because PD-1 and PD-L1 are also expressed on anti-tumor T cells, the utilization with PD-1 or PD-L1 MAbs usually accompanied with undesirable side effect including depletion of the very lymphocytes they are intended to activate. Taken together, development of small molecules or low-molecular weight protein drugs are still in urgent need even with the existence of so many antibody drugs under development or in the market.In addition, there are also very reasons that the development of small molecules or low-molecular weight protein drugs targeting PD-1/PD-L1 signal are in urgent need to achieve a more efficient treatment. First, the efficacy of PD-1/PD-L1 blockade strongly depends on the molecular accession of antibody with PD-1 or PD-L1. However, accession of PD-1 or PD-L1 molecules in the tumor microenvironment by the penetrated MAbs is less efficient because of the large size of the MAbs and the complexity of tumor microenvironments, which further limits their functional potential.39–41 Based on the complex structure of mPD-1/hPD-L1, Roy Maute et al.42 designed a yeast-surface display system for a directed evolution to generate high-affinity PD-1 that antagonizes PD-L1. 22 amino acid residues of PD-1 contributing to the contacts with PD-L1 were selected for randomization. Two-generations of yeast-surface display library evolution were conducted which resulted in a high-affinity consensus hPD-1 with enhanced binding affinities (Kd) of ∼100 pm with hPD-L1 compared with the mild binding affinities of a Kd value of 3.8 μm for wild type hPD-1. In contrast to anti-PD-L1 MAbs, high-affinity PD-1 enabled superior tumor penetration without inducing depletion of peripheral effector T cells. In addition, the high-affinity PD-1 also showed superior tumor suppression efficacy compared with antibodies in mouse colon carcinoma model. Moreover, a small molecule drug developed by Curis, Inc. , CA-170  checkpoint antagonist), has entered into Phase I clinical trial in patients with advanced tumors and lymphomas (NCT02812875).Complex structure of PD-1/PD-L1 has brought promising perspectives to structure-based drug design to interrupt the PD-1/PD-L1 interaction. It is now clear how the PD-1 interact with PD-L1 since murine PD-1 (mPD-1)/hPD-L1, mPD-1/mPD-L2 and hPD-1/hPD-L1 complex structures were resolved.The recent report on pembrolizumab/hPD-1 and avelumab/hPD-L1 complex structures have enabled the scientists to see how the checkpoint blockade MAbs interact with the corresponding targets and how this would compete the binding with PD-1/PD-L1. All this information would shed light on the development of the next-generation MAbs or small inhibitory molecules. However, additional therapeutic antibody/PD-1 (or PD-L1) complex structures are still needed to draw a clearer map of how therapeutic MAbs work. Structural immunology has now helped the scientists to see what the immune checkpoint therapeutic MAbs look like and how they work, it is now the responsibility of researchers from both basic science and industry to build more efficient therapeutics to bedside.


Processing and manipulation of highly conductive pristine graphene in large quantities are still major challenges in the practical application of graphene for electric device. In the present study, we report the liquid-phase exfoliation of graphite in toluene using well-defined poly(3-hexylthiophene) (P3HT) to produce a P3HT/graphene composite. We synthesize and use regioregular P3HT with controlled molecular weights as conductive dispersants for graphene. Simple ultrasonication of graphite flakes with the P3HT successfully produces single-layer and few-layer graphene sheets dispersed in toluene. The produced P3HT/graphene composite can be used as conductive graphene ink, indicating that the P3HT/graphene composite has high electrical conductivity owing to the high conductivity of P3HT and graphene. The P3HT/graphene composite also works as an oxidation-resistant and conductive film for a copper substrate, which is due to the high gas-barrier property of graphene. Graphene is one of the most interesting nanomaterials owing to its unique properties, such as high electrical conductivity, high mechanical toughness, high gas-barrier performance, and unique optical properties124810111213141516Poly(3-hexylthiophene) (P3HT) has attracted attention in fields such as material science and electronics owing to its high electrical conductivity and its processability192021222325262(PPh3)IPr) were purchased from Tokyo Chemical Industries, Ltd. . 2,2,6,6-Tetramethylpiperidinylmagnesium chloride lithium chloride complex (TMPMgCl·LiCl) solution was purchased from Sigma . Other chemicals were purchased from Wako PureChemical Industries, Ltd. . The water used was high-quality deionized water  produced using a Milli-Q Advantage A100 system equipped with an Elix UV 3 system .Graphite flakes and copper-plated divinylbenzene microparticles (CuMPs) were provided by Sekisui Chemical Co., Ltd. . 2-Bromo-3-hexylthiophene and triphenylphosphine nickel(II) dichloride (NiCl2(PPh3)IPr  were successively added. The resulting mixture was stirred at 25 °C for 1 h. Hydrochloric acid  and methanol (20 mL) were added to form a precipitate. The mixture was filtered and the residue was washed with methanol and n-hexane repeatedly to obtain a dark purple solid, which was dried under reduced pressure to yield 144 mg of poly(3-hexylthiophene) (P3HT) (66%). Molecular weights and the molecular weight distribution of P3HT were estimated by SEC analysis (eluent: CHCl3) using polystyrene as the molecular weight standard. For example, SEC analysis showed Mn = 6000, and Mw/Mn = 1.10. The regioregularity was estimated by 1H NMR analysis  at the δ 2.80 (H-T) and δ 2.60 (H-H) signals (H-T regioregularity = 97%).P3HT was synthesized by the slightly modified procedure previously reported by us2526Mw/Mn = 1.6) was synthesized according to a previous reportRegio-irregular P3HT  at 1000 × g for 20 min to remove the unexfoliated particles and thick flakes of graphite. Then, the supernatant was collected as the graphene dispersion.Graphite flakes were ultrasonicated  with P3HT in toluene at 80 °C for 30 min, followed by incubation at 30 °C for 15 min. The concentration of graphite flakes was 0.33 mg mLMn = 6,000) or SDS, were filtrated through filter paper that were 8 mm in diameter and 4 μm of pore size . P3HT/graphene (or SDS/graphene) on the filter paper was dried at 100 °C for 5 min to ensure complete solvent evaporation.P3HT/graphene composite film-papers were prepared by vaccum filtration. Graphene dispersions (3 ml) containing 42 μg graphene, which were prepared using P3HT (2SO4 aqueous solution (0.1 M) for 10 min at 25 °C to remove copper oxide from the surfaces. Then, they were rinsed with excess amounts of water and ethanol three times each. P3HT- and P3HT/graphene-coated CuMPs were prepared by dip-coating the rinsed CuMPs (20 mg) in P3HT/graphene dispersion (400 μL) for 1.5 h at 40 °C, followed by the removal of the supernatant by vaccum evaporation overnight to dry the P3HT/graphene-coated CuMPs.The CuMPs were rinsed with HUV/Vis absorption spectra of the dispersions (200–900 nm) were recorded using a double-beam spectrophotometer . Since P3HT did not exhibit an absorbance around 660 nm, absorption measurements at 660 nm allowed us to evaluate the concentration of dispersed graphene in toluene.Raman spectroscopy measurements were performed on a Nano-Raman system WITec alpha300R using a laser excitation at 532 nm  at 3 mW.TEM images were obtained using a JEOL 2100 F transmission electron microscope operated at 200 kV. A drop of graphene dispersion was placed on a copper grid covered with elastic carbon films manufactured by Okenshoji Co., Ltd. and dried under vaccum at ambient temperature.−1. SEM observations were performed using a JEOL JSM-7500F scanning electron microscope.AFM images were obtained in a tapping mode using Hitachi Nanonavi E-sweep with cantilevers at 17,000 N mElectrical resistance measurements were performed using a four-point-probe ohmmeter  for P3HT/graphene composite paper films and a digital multimeter  for CuMPs.Mn ranging from 6,000 to 75,000 with a very narrow distribution of molecular weights  was the most effective in dispersing graphene. P3HT with molecular weights of more than 40,000 resulted in low graphene concentrations in the dispersion, which might be related to the low solubility of the high-molecular-weight P3HT in toluene18We evaluated the effect of the Mn 6,000 was used and the P3HT/graphite was 1.0, the graphene concentration in toluene was 14 μg mL−1, in which the yield of graphene was 3.5%.The effect of the P3HT/graphite ratio on dispersing graphene was also investigated . As the Mn 7000 and Mw/Mn = 1.6) at a P3HT/graphite ratio of 1.0 to exfoliate graphite and to disperse graphene in toluene. Interestingly, there observed negligible absorbance at 660 nm , an atomic force microscope (AFM), and Raman spectroscopy. TEM images of the P3HT/graphene dispersion are shown in −1), G band (1584 cm−1), and 2D band (2680 cm−1) of the analytes32ID/IG ratio of 0.63, which was almost the same as that of the graphite flakes. These measurements indicate that defects in the graphene sheet did not occur during the P3HT-induced exfoliation of the graphite and graphene dispersion.The above investigations suggest that P3HT works as a dispersant for graphene in toluene. Defects in graphene often occur during the exfoliation of graphite and dispersion of graphene in solvents30−1, which was much smaller than that of the SDS/graphene composite (31.6 ± 3.0 kΩsq−1). The resistance of the P3HT/graphene-composite was comparable with those of graphene films with other dispersants111536123539To investigate the electrical property of the P3HT/graphene composite, P3HT/graphene and sodium dodecyl sulfate (SDS)/graphene composites on filter paper were prepared by vacuum filtration of the graphene dispersion 34. The sOne of the unique properties of graphene is its molecular barrier property, which prevents gas permeation34142−1  for the preparation of conductive polymer/graphene composite. Sci. Rep.7, 39937; doi: 10.1038/srep39937 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


PD-L1 promoter assays, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that IFNγ-inducible PD-L1 overexpression is primarily mediated by the putative IRF-1α site of the PD-L1 promoter in EBV (+) SNU-719 cells. Moreover, EBNA1 knockdown reduced both constitutive and IFNγ-inducible PD-L1 promoter activity by decreasing the transcript and protein levels of JAK2 and subsequently STAT1/IRF-1/PD-L1 signaling. EBNA1 is suggested to be moderately enhance both constitutive and IFNγ-inducible PD-L1 expression in EBV (+) GC cells. Thus, the signaling proteins and EBNA1 that regulate PD-L1 expression are potential therapeutic targets in EBV (+) GC.Programmed death-ligand 1 (PD-L1) acts as an immune checkpoint inhibitor in various cancers. PD-L1 is known to be more frequently expressed in EBV (+) gastric cancer (GC). However, the mechanisms underlying the regulation of PD-L1 expression in EBV (+) GC remain unclear. We investigated the basal and inducible PD-L1 expressions in GC cells. PD-L1 expression was upregulated upon treatment with IFNγ in both EBV (−) and EBV (+) GC cells. Upon stimulation with the same concentration of IFNγ for 24 h, EBV (+) SNU-719 cells showed dramatically higher PD-L1 expression levels by activating JAK2/STAT1/IRF-1 signaling than those of EBV (−) AGS cells.  PD-L1 is upregulated in various epithelial and lymphoid tumors, including gastric cancer (GC)5. Anti-PD-L1 or anti-PD-1 antibody has recently been used as an immune checkpoint inhibitor6. Although the functional relationships between PD-L1 in cancer cells and PD-1 in cytotoxic T cells have been established9, the mechanisms by which PD-L1 expression is regulated in cancer cells remains unclear.Programmed death ligand-1 (PD-L1), known as B7-H1 or CD274, is a glycoprotein of the B7 superfamily that is expressed on the cell surface of various tumor cells, as well as in lymphocytes, macrophages, and dendritic cells. PD-L1 functions as a PD-1 ligand, which binds to PD-1 on the cytotoxic T lymphocytes to inhibit the immune responses10. EBV (+) GC accounts for about 10% of all GCs and PD-L1 overexpression is observed in over 50% of EBV (+) GCs. The prognostic significance of PD-L1 overexpression in GC patients remains controversial and has not yet been clearly defined13. Amplification of the 9p24.1 locus, which leads to PD-L1 overexpression, is observed in approximately 11–15% of EBV (+) GC cases13. However, other mechanisms responsible for PD-L1 upregulation aside from PD-L1 gene amplification in EBV (+) GC have not been explored.GCs have recently been categorized in The Cancer Genome Atlas (TCGA) as Epstein-Barr virus (EBV)-positive, microsatellite instable (MSI), chromosomal instable (CIN), and genomically stable (GS)15. EBNA1 is a transcriptional factor in viral DNA replication and maintains the constant copy number of EBV genomes during cell division16. EBNA1 regulates the expression of other EBV genes and host cellular genes17. EBNA1 has a cis-acting immune evasive mechanism. The glycine-alanine repeat domain (GAr) in EBNA1 inhibits the translation of its own mRNA, thereby minimizing the production of antigenic peptides that activate the major histocompatibility complex (MHC) class I pathway18. Moreover, EBNA1 has been shown to increase STAT1 expression in three different carcinoma cell lines20. EBNA1 enhances phosphorylation and nuclear localization of STAT1 in response to IFNγ20.All EBV (+) GCs (EBV latency I or II) express EBV nuclear antigen 1 (EBNA1), EBERs, BARTs, and BART miRNAs. Approximately 50% of EBV (+) GC cases express LMP2A8. IFNγ also regulates IRF-1 expression in lung cancer cells7. However, the roles of IFNγ and EBNA1 in PD-L1 expression of EBV (+) GC remain to be determined.IFNγ is a cytokine secreted by tumor–infiltrating T cells and induce PD-L1 expression by stimulating the JAK/STAT signaling pathway in myeloid leukemia cellsTherefore, we investigated whether IFNγ induces PD-L1 expression and compared the mechanisms by which EBV influences IFNγ-induced PD-L1 expression in EBV (+) GC and EBV (−) GC cells.PD-L1 gene in about 11–15% of EBV (+) GCs13. By contrast, in this study, all GC cell lines, except for MKN-28, had normal copy numbers of the PD-L1 gene; however, AGS and SNU-719 cells showed high PD-L1 levels  cell lines, namely, AGS, MKN-1, MKN-28, and SNU-601, and in three EBV (+) cell lines, namely, SNU-719, YCCEL1, and NCC-24. PD-L1 expression was compared with that of EBNA1 in three EBV (+) GC cell lines. PD-L1 was constitutively overexpressed in both mRNA and protein levels in the EBV (−) AGS and MKN-28 cell lines, as well as in the EBV (+) SNU-719 cell line Fig. . SNU-719els Fig. . Paradoxels Fig. . These r22. All GC cell lines constitutively expressed IFNGR2, JAK2, STAT1, IRF-1, and PD-L1 mRNAs at varying levels, but IFNGR1 expression was relatively low or absent compared to IFNGR2  GCs Fig. , we exam8 and induces PD-L1 expression through JAK/STAT signaling in lung cancer cells7. Furthermore, immunohistochemical analysis of EBV (+) GC tissues showed that PD-L1 was particularly strongly expressed in EBV (+) GC cells confronting infiltrating lymphocytes  GC and five CIN–type GC tissues showed that IFNγ and IFNT1 were significantly enriched in EBV (+) GC cases than in CIN–type GC Fig. . In addites Fig. . TherefoTo compare the expression of PD-L1 by IFNγ in EBV (−) and EBV (+) GC cells, the appropriate concentration and duration of IFNγ treatments was first determined. PD-L1 expression in GC cells under the optimal IFNγ treatment conditions was then assayed Fig. . PD-L1 m21 and our current GSEA results suggest that IFNγ induces PD-L1 expression via activation of JAK2/STAT1/IRF-1 signaling in EBV (+) GC cells. To verify these results, EBNA1, PD-L1, and JAK2/STAT1/IRF-1 signaling pathway were examined by RT-PCR, qRT-PCR, and immunoblotting. Alteration of these genes were also analyzed using the OncoPrint from TCGA EBV (+) GC tissue data. IFNγ induced PD-L1 upregulation at both the mRNA and protein levels via phosphorylation of JAK2 and STAT1 and subsequent increase of IRF-1 expression in EBV (+) SNU-719 cells  GC tissues, respectively. All EBV (+) GC tissues showed almost the same gene alterations or transcriptional upregulation patterns. Interestingly, this phenomenon was also observed in the EBV (+) GC cases without ion Fig. .PD-L1 promoter were significantly decreased in IFNγ-stimulated SNU-719 cells pretreated with AZD1480 or fludarabine, compared to only IFNγ-stimulated SNU-719 cells  and fludarabine (a STAT1 inhibitor that depletes STAT1 mRNA and protein). The luciferase activities of lls Fig. . The JAKlls Fig. . AZD1480lls Fig. , whereaslls Fig. . These rPD-L1 that are regulated by IFNγ and EBV, we measured PD-L1 promoter activity using the reporter constructs in EBV (−) and EBV (+) IFNγ–stimulated and unstimulated GC cells  was induced 1.3 times higher in IFNγ-treated EBV (−) AGS cells than in the untreated control  SNU-719 cells  GC cells.To identify the specific IRF-1 binding at the IRF-1α site of the bes Fig. . Additiobes Fig. . These rPD-L1 promoter activity in SNU-719 cells with or without IFNγ treatment after EBNA1 knockdown with siRNA. PD-L1 full promoter activity was significantly decreased in SNU-719 cells with EBNA1 knockdown compared to mock and scramble siRNA-transfected control SNU-719 cells. However, activities of the three IRF-1-deleted promoters were not significantly decreased  and EBV (+) GC cells, notably, EBV (+) GC cells show significantly higher PD-L1 expression by activating JAK2/STAT1/IRF-1 signaling compared to EBV (−) GC cells. The IFNγ-induced IRF-1 was shown to bind to the IRF-1α DNA sequence of the PD-L1 promoter in EBV (+) GC cells. EBNA1 knockdown reduces the constitutive and IFNγ-inducible PD-L1 promoter activity by decreasing the transcript and protein levels of JAK2 and subsequently the STAT1/IRF-1/PD-L1 signaling.In this study, we revealed that constitutive PD-L1 expression is correlated with EBNA1 expression in EBV (+) GC cell lines without 15. EBNA1 is a viral nuclear protein essential for the maintenance of the EBV genome and has a cis-acting mechanism to prevent immune recognition of EBNA1 expressing tumor cells by cytotoxic T lymphocytes23. The size of EBNA1 protein in EBV (+) cell lines was dependent on the GAr size of EBNA118. Interestingly, this study showed that the smaller the size of EBNA1, more specifically the GAr size, the higher the expression of EBNA1 and PD-L1. Differences in EBNA1 expression between EBV (+) GC cell lines do not appear to be due to differences in EBV copy number among EBV (+) GC cell lines. Despite SNU-719 and YCCEL1 cell lines have similar numbers of EBV copies per cell24, EBNA1 expression levels were different in this study. The GAr of EBNA1 suppresses the translation of EBNA1 mRNA, preventing the antigen peptide presentation to the MHC class I pathway, and eventually, play a role of immune evasion25. However, there has not been reported that the size of EBNA1 GAr is directly inversely proportional to the transcription and protein expression of EBNA1 mRNA and even to the expression level of PD-L1, rather than the translation of EBNA1 mRNA25. Thus, this direct association with the size of EBNA1 GAr and EBNA1 transcription requires further investigation. EBNA1 proteins of SNU-719 and YCCEL1 cells contain 589 and 625 amino acids, respectively. Compared to YCCEL1 cells, SNU-719 cells partially deleted the EBNA1 GAr and EBNA1 protein showed about 94.1% amino acid identity between two cell lines  GC cell lines. Thus, our study indicates that EBNA1 may play a role in constitutive and IFNγ-inducible PD-L1 overexpression.This study found that the levels of EBNA1 expression are correlated with constitutive and inducible PD-L1 levels in the three EBV (+) GC cell lines. EBV (+) GC is known to express only two latent viral proteins, EBNA1 and LMP2A. Among these, EBNA1 is expressed in almost all patients with EBV (+) GC, whereas LMP2A is detected in approximately 50% of patients with EBV (+) GC27, the detailed mechanisms by which IFNγ induces PD-L1 expression in EBV (+) GC cells remain unclear. The GSEA using TCGA GC tissue data suggested that IFNγ could activate the JAK/STAT signaling pathway in EBV (+) GC. In addition, immunohistochemical analysis using EBV (+) GC tissues revealed that PD-L1 was strongly expressed throughout EBV (+) GC cells infiltrating with many T lymphocytes. Thus, it was hypothesized that IFNγ released from tumor-infiltrating T lymphocytes could activate the JAK/STAT signaling pathway in EBV (+) GC cells. In this study, we displayed that IFNγ stimulation promotes PD-L1 overexpression by activating JAK2/STAT1/IRF-1 signaling in EBV (+) GC. Furthermore, IFNγ-induced IRF-1 was found to bind to the IRF-1α DNA sequence of the PD-L1 promoter. Activation of this PD-L1 signaling pathway was reaffirmed in EBV (+) GC because the IFNγ-stimulated JAK2/STAT1/IRF-1/PD-L1 signaling was suppressed with AZD1480 (an ATP-competitive JAK2 inhibitor) and fludarabine (a STAT1 inhibitor which causes a specific depletion of STAT1 protein and mRNA), respectively.We found that IFNγ-induced PD-L1 overexpression is mediated by activation of JAK2/STAT1/IRF-1 signaling pathway in EBV (+) GC. Although IFNγ has been previously demonstrated to increase PD-L1 expression in GC cellsPD-L1 promoter activity in EBV (+) GC cells. EBNA1 induces transcriptional upregulation of JAK2, leading to constitutive and IFNγ-induced PD-L1 expression. In contrast, LMP2A did not upregulate PD-L1 expression and PD-L1 promoter activity in constitutive and IFNγ-induced EBV (+) GC cells, based on LMP2A knockdown experiments  SNU-719 cells, IFNγ-stimulation on EBV (−) AGS cells seems to activate the STAT1/IRF-1/PD-L1 signaling without going through the phosphorylation step of JAK2. Moreover, when EBNAl-transfected AGS cells were stimulated with IFNγ, AGS cells appeared to have only the effect of IFNγ, but there was no effect of ectopic EBNAl expression (data not shown). Additional experiments such as ectopic EBNA1 expression in EBV (−) GC cell lines without PD-L1 expression, including MKN-1 and SNU-601 cells, are required.Under basal conditions, EBV (−) AGS cells already express high levels of PD-L1 protein, most likely due to an intrinsic oncogenic activationPD-L1 transcription by binding to the IRF-1α site of the PD-L1 promoter. In addition, EBNA1 partially enhances IFNγ-mediated PD-L1 expression through upregulation and activation of JAK2. Recently, anti-PD-L1 and anti-PD-1 antibodies to the PD-L1/PD-1 axis have been shown to be encouraging as immunotherapies in a variety of cancers34. Given that PD-L1 is strongly expressed upon IFNγ stimulation in EBV (+) GC cells, the immune evasion of EBV (+) GC cells could be regulated by inhibiting JAK2/STAT1/IRF-1 signaling as well as by controlling EBNA1 expression.In summary, our results indicate that IFNγ-mediated PD-L1 overexpression is regulated by the JAK2/STAT1/IRF-1 signaling pathway in EBV (+) GC cells. IRF-1 directly regulates 2. Informed consent was obtained from all patients and the experimental protocol was reviewed and approved by the Tissue Ethical Committee of Korea University Ansan Hospital for the use of tissue specimens (No. 2016-004). The method was carried out in accordance with the committee’s approved guidelines. Paraffin-embedded GC and non-malignant gastric tissue specimens were used for immunohistochemistry.The gastric cancer cell line AGS was obtained from the American Type Culture Collection . EBV (−) GC cell lines  and EBV (+) GC cell lines (SNU-719 and NCC-24) were obtained from the Korean Cell Line Bank . The six cell lines were cultured in RPMI1640 medium. The EBV (+) YCCEL1 GC cell line was a kind gift from Dr. SY Rha from the Yonsei University College of Medicine. YCCEL1 cells were cultured in minimum essential medium (MEM). Media were supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO® Green Gene Expression Assays (Thermo Fisher Scientific). Primer sequences are summarized in Supplementary Table β-actin was amplified in the same reaction to serve as an internal control for normalization. Fold changes in gene expression were measured using the comparative threshold cycle method (ΔΔCt).Total RNA was extracted from cell lines using TRIzol  and subjected to reverse transcription. qRT-PCR was performed in triplicate using a 7500 Real-Time PCR System and Power SYBRβ-actin  antibodies. Blots were incubated with horseradish peroxidase-conjugated secondary antibodies  and visualized using ECL . Immunohistochemical analysis was performed using the Leica BOND-MAX autostainer and Leica Refine detection kit  using primary PD-L1 antibody .Protein lysates were subjected to electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Blots were incubated using primary anti-PD-L1, JAK2, pJAK2 (Y1007/1008), STAT1, pSTAT1 (Y701) , IRF-1, EBNA1, and PD-L1 DNA copy number was performed in triplicate using a 7500 Real-Time PCR System (Thermo Fisher Scientific) and TaqMan®PD-L1 Copy Number Assays  with Reference Assay RNase P (Thermo Fisher Scientific). Data were analyzed using CopyCaller® Software v2.0 (Thermo Fisher Scientific). Average value from five normal gastric tissues was used as a normal control.qRT-PCR for PD-L1 promoter were constructed using pGL4.15 vector. The primer sequences are summarized in Supplementary Table PD-L1 promoter constructs using Lipofectamine 2000® (Life Technologies). After 4 h, the culture medium was replaced with complete medium with or without IFNγ . At 24 h after transfection, the cells were washed and lysed. Cell lysates were analyzed for luciferase activity using a dual luciferase assay kit (Promega) and an EnSpire multimode reader . The Renilla luciferase construct, pRL-TK, was co-transfected as an internal control.The luciferase reporters of 6 cells and treated with 5, 10, 20 μM of AZD1480 or fludarabine for 2 h, followed by treatment with 10 ng/mL IFNγ for 24 h. The cells harvested and analyzed for mRNA and protein expressions. DMSO was included in each plate as a negative control.AZD1480 and fludarabine were purchased from selleckchem . For the signaling inhibition experiments, AZD1480 and fludarabine dissolved in 100% dimethyl sulfoxide  were prepared in a 10 mM stock and stored at −70 °C. SNU-719 cells were seeded in 6-well plates at a density of 1 × 10PD-L1 promoter in SNU719 and AGS cells were analyzed using a ChIP assay kit . Cells were treated with formaldehyde for 10 min at 37 °C, incubated in lysis buffer, and sonicated to fragment the chromatin. The crosslinked protein-DNA complexes were immunoprecipitated with anti- IRF-1 antibody (Santa Cruz Biotechnology). Anti-histone 3  antibody and Rabbit IgG  were used as positive and negative controls, respectively. Cross-links were reversed after performing a pull-down assay on antibody-bound complexes with protein A-agarose/salmon sperm DNA (Millipore). DNA was recovered by phenol/chloroform extraction and ethanol precipitation and used as a template for subsequent PCR amplification. The primer sequences for PCR are summarized in Supplementary Table Physical associations between the IRF-1 and IRF-1 binding sites of the Nuclear extracts were obtained using a nuclear extract kit  and were incubated with a biotin-labeled probe for 20 min in binding buffer (Active Motif). For supershift analysis, extracts were incubated with anti-IRF-1 antibody (Santa Cruz Biotechnology) and control antibody . Reaction mixtures were separated on 6% acrylamide gels. Wild-type and mutant probe sequences are listed in Supplementary Table ® RNAiMAX (Thermo Fisher Scientific). The EBNA1 siRNA sequences are summarized in Supplementary Table ® medium (Thermo Fisher Scientific) and combined afterwards. The resulting mixtures were incubated for 5 min and added dropwise to each culture well containing 1 mL of Opti-MEM® medium. After 4 h, the medium was replaced with the fresh complete RPMI1640 medium. Cells were cultivated for 48 or 72 h, washed in phosphate-buffered saline , and harvested.SNU-719 cells were grown to 70% confluence for 24 h, serum-starved for 30 min, and transiently transfected using Lipofectamine36. RNA-sequencing data were downloaded and preprocessed using TCGAbiolinks and annotated with Entrez ID. Differentially expressed genes between EBV (+) and EBV (−) CIN-type GCs were identified using the limma-voom with adjustment for false discovery rate and hierarchical clustering was performed using the heatmap.2 function of the gplots. Enrichment for differential regulated gene sets was calculated using EGSEA. Hallmark gene sets were downloaded from the MSigDB database (http://software.broadinstitute.org/gsea/msigdb). Fold changes of gene expression were projected onto Kyoto Encyclopedia of Genes and Genomes (KEGG) maps (http://www.genome.jp/kegg/)37. OncoPrint analysis of GC TCGA was performed on the cBioPortal for Cancer Genomics.GSEA was performed using Bioconductor R packages, including TCGAbiolinks and Ensemble of GSEA (EGSEA)t-test and one-way analysis of variance (ANOVA). Statistical significance was considered at p-value < 0.05. All statistical analyses were performed with SPSS for Windows 10.0 .Experiments were repeated at least three times. Mean values were compared using two-tailed Student’s Supplementary information


In this study, we propose a tilted fiber Bragg grating (TFBG) humidity sensor fabricated using the phase mask method to produce a TFBG that was then etched with five different diameters of 20, 35, 50, 55 and 60 μm, after which piezoelectric inkjet technology was used to coat the grating with graphene oxide. According to the experimental results, the diameter of 20 μm yielded the best sensitivity. In addition, the experimental results showed that the wavelength sensitivity was −0.01 nm/%RH and the linearity was 0.996. Furthermore, the measurement results showed that when the relative humidity was increased, the refractive index of the sensor was decreased, meaning that the TFBG cladding mode spectrum wavelength was shifted. Therefore, the proposed graphene oxide film TFBG humidity sensor has good potential to be an effective relative humidity monitor. Furthermore, fiber optic sensors for biomedical and home security purposes have also been developed in recent years. The mechanism by which such sensors provide measurements is very dependent on changes in the refractive index, such as in the case of LPG gas sensor measurements of CO2  and U-sh2 ,5,6,7,8,2 ,4, and R2 . The pre2 , a long 2 , and a h2 . HoweverThe proposed sensor utilizes a tilted fiber Bragg grating (TFBG) to achieve the humidity sensing function because TFBG can effectively measure the surrounding refractive index. This is because when the refractive index changes, the wavelength of the cladding mode of the TFBG will shift in a way that corresponds to the ambient humidity value.Humidity sensors are often used in conjunction with cooled computers, heat treating furnaces, smelting furnaces, clean room controls, and dryers, as well as in applications relating to drug administration, food preservation, and climatic measurements. However, while some harsh environments, such as high temperature environments or environments with high levels of electromagnetic waves, will have an impact on the sensitivity of previously produced humidity sensors, optical fiber humidity sensors could potentially overcome those environmental challenges.In TFBG, there is a certain tilt angle between the grating plane and the fiber, so the transmission spectrum possesses many resonances , resultiIn 1996, Erdogan and Sipe , using aTherefore, this study proposes for the first time, to the best of our knowledge, a small diameter TFBG sensor (20 μm) with a graphene oxide coating for humidity sensing. The novelty of the proposed TFBG sensor is twofold: first, the sensitivity of the sensor is increased by reducing the fiber diameters via etching; second, we provide new coating technology used the piezoelectric inkjet can provide a uniform graphene oxide coating and fast processing, allowing it to be used as a humidity sensor.Mode fiber coupling between the wave vectors of all vector fields occurs along the fiber axis to form a uniform axial conduction mode structure that is defined by a flat phase plane perpendicular to the guide shaft, as shown in The Bragg resonance conditions described above are produced by the coupling of mode-forward propagating core modes and back-propagating core modes. For TFBG, the inclination angle of the grating surface and the axis of the fiber, as well as the grating period along the fiber axis, can be modified according to the following Equation :(2)Λg=ΛcSubstituting Equation (2) into Equation (1), yields the following:Due to the presence of the tilted angle, part of the light propagating in a forward direction through the core mode will be coupled to the cladding mode of the backward propagation, and the resonant wavelength of the cladding mode will be determined by the following equation :(4)λCl,iDue to the interaction between the cladding mode and the graphene oxide, the resonance of the cladding mode changes as the graphene oxide adsorption process proceeds, because the effective refractive index of the graphene oxide film changes as the water molecules are adsorbed. As can be seen from Equation (4), when the humidity increases, the refractive index changes, causing the wavelength to be shifted, and through this phenomenon, the changes in the TFBG sensor effectively correspond to the relative humidity changes.In this study, the TFBG humidity sensor was fabricated using single-mode fiber (SMF-28), the phase mask method, and piezoelectric inkjet technology. Using a wire stripper, 3 cm of the protective middle layer was stripped from optical fiber, after which the fiber was wiped clean with alcohol and then placed on a 3-axis micro-platform. Next, the mask platform was rotated 10 degrees and excimer laser  irradiation was applied through phase masks  to produce the grating formation. An optical spectrum analyzer was then utilized to confirm the generation of TFBG in the core of the spectrum structure, as shown in The TFBG was etched to 20 μm, and then piezoelectric inkjet technology  was used to spray an aqueous solution of graphene oxide onto the surface of the optical fiber. The coated fiber was then heated to 100 degrees on a baking plate to induce evaporation, allowing the graphene to become more securely attached to the optical fiber surface, as shown in The fiber was placed in a humidity control box. A light source  was attached to one end of the box, while an optical spectrum analyzer was attached to the other. humidification control range of 20–80% RH, temperature controller setting 27 °C, standard humidity sensor for compared to our TFBG GO humidity sensor, humidifier to increase the environment relative humidity, dehumidification is the dry air into the (a flow rate of 10 L/min) humidity control Box to reduce humidity, as shown in In this study, the TFBG-coated graphene oxide film coated on SMF-28 single-mode photosensitive fiber was etched with five different diameters of 20, 35, 50, 55 and 60 μm, and the relative humidity was measured at a constant temperature of 27 °C and a humidity range of 20% to 80% RH.In this study, piezoelectric inkjet technology was used to coat graphene oxide film on optical fiber. The thickness of the coated graphene oxide, as measured by scanning electron microscope (SEM) imaging, was about 1 μm. Enlargement of the SEM image shown in 2O molecules (H2O = H+ + OH−) and the C−OH and C=O groups of the GO. Such an increase in interlayer spacing is also consistent with previous studies on the interlayer spacing of graphene oxide as a function of relative humidity. When the humidity in the air increases, more water molecules are adsorbed on the graphene oxide, and the water molecules on the graphene layer will, in turn, change the gap between the graphene and the fiber. In a previous study of graphene oxide thin films , these cWe conducted an initial temperature experiment to verify the effect of temperature on the TFBG sensor, with the results being shown in The strength of the transmission, which is less strong without the etching, becomes stronger after the etching, resulting in the wavelength being shifted from a short wavelength to a long wavelength. After etching, the cladding mode is significantly enhanced, which improves the ability of the fiber to measure the refraction index, as shown in The TFBG coating graphene oxide humidity sensor has the best sensitivity in the number of diameters. In this paper, the sensitivity of the sensor to humidity changes was measured with different diameters of 20, 35, 50 55 and 60 μm. As can be seen from the discussion in As shown in At a diameter of 20 μm, the sensitivity was −0.01 nm/% RH, and the linearity was 0.996. At a diameter of 35 μm, the sensitivity was −0.00425 nm/% RH, and the linearity was 0.990. At a diameter of 50 μm, the sensitivity was −0.0031 nm/% RH, and the linearity was 0.985. At a diameter of 55 μm, the sensitivity was −0.0022 nm/% RH, and the linearity was 0.995. At a diameter of 60 μm, the sensitivity was −0.0021 nm/% RH, and the linearity was 0.996. Through the experimental results detailed above, we found that the diameter of 20 μm had the best wavelength sensitivity, as shown in The wavelength sensitivity in the first cycle was −0.01 nm/% RH, and the linearity was 0.996. The wavelength sensitivity in the second cycle was −0.01 nm/% RH, and the linearity was 0.996. The wavelength sensitivity in the third cycle was −0.01 nm/% RH, and the linearity was 0.996. The optimal wavelength sensitivity was −0.01 nm/% RH, and the linearity was 0.996. From the results for the three cycles, it can be seen that the sensitivity and linearity were the best in the third cycle. From the error bars shown in After three cycles of testing, we can see that the relative humidity measured by the TFBG graphene oxide sensor was close to the relative humidity measured by the standard humidity sensor, which shows that the TFBG sensor with graphene oxide coating has the same accuracy for humidity sensing despite being cheaper.From the analysis results detailed in We have provided the preliminary experimental data for a sensor stability and durability test. According to the experimental results, the humidity sensitivity measured at 14 days was still quite close to the initial sensitivity, as shown in In this study, a TFBG humidity sensor was fabricated by coating TFBG with a graphene oxide film. The measurement results showed that the measurement sensitivity of the humidity was 4.695 times higher than 0.00213 nm/% RH , 5 times


The recent FDA approval of two drugs, pirfenidone and nintedanib, for the treatment of idiopathic pulmonary fibrosis (IPF) has fueled interest in the development of additional drugs to treat the disease or its major clinical complications including cough and acute exacerbations. Since 2015, there are at least a dozen active interventional studies that are testing the efficacy of novel pharmacotherapies, exercise or stem cells in modifying the disease process in IPF. Additionally, there are combinatorial studies evaluating the effectiveness of pirfenidone or nintedanib in combination with other agents. However, there remains an urgent need for clinical trials to prospectively evaluate the efficacy of existing drugs with promising retrospective data, such as proton pump inhibitors (PPIs), in IPF. Several retrospective cohorts have provided tantalizing data supporting the beneficial effect of PPIs in patients with well-defined IPF. This review provides the general outlook of pharmacotherapies in IPF, and highlights preclinical and retrospective clinical data to make a case for randomized controlled clinical trials of PPIs in IPF. The efficacy of pirfenidone and nintedanib in reducing disease progression including better preservation of lung function, exercise tolerance and progression-free survival has led to concurrent FDA approval of the two drugs for IPF . AlthougTable 1). Some of the emerging effects of PPIs include regulation of oxidative stress through scavenging reactive oxygen species (ROS) and activation of endogenous antioxidant mechanisms including heme oxygenase 1 (HO1) (4)-induced liver fibrosis . Omeprazole (Prilosec), esomeprazole (Nexium), lansoprazole (Prevacid), and rabeprazole (Aciphex) are among the most commonly used PPIs. Several lines of preclinical research indicate that PPIs possess biological activities that extend beyond gastric acid suppression ( 1 (HO1) . The lat 1 (HO1) . Consist 1 (HO1) . In addi 1 (HO1) . Similar 1 (HO1) . Further 1 (HO1) . Recentl 1 (HO1) . The antfibrosis . Based ofibrosis . Overallp < 0.01). Accordingly, the hazard ratio (HR) in the treatment arm was intriguingly reduced to 0.5 (CO) and reduced composite physiologic index (CPI); a validated measure of disease severity in IPF  including hypersecretion of gastric juice and/or clinical symptoms such as heartburn, nausea, chest pain and regurgitation . In addid to 0.5 . In 2012y in IPF . Surprisy in IPF . The prey in IPF .N-acetylcysteine) have failed to meet their endpoints , the use of PPIs among the patients who were designated in the “placebo” arm of these 3 studies was significantly associated with lower loss in lung function  and fewer acute exacerbations. In addition, there was a trend toward fewer all-cause hospitalizations and all-cause mortality in the patients who received GER medications (over 90% of whom on PPIs) compared to these without the medications.In 2013, the IPF Clinical Research Network (IPFnet) group analyzed three ILD databases containing 242 patients who participated in three large randomized controlled trials  . Althougp < 0.01) compared to these who were only on standard of care. In a subgroup analysis of IPF patients with no symptoms of GERD, the use of PPIs was also associated with significantly longer survival time (p = 0.009)  . In the Post hoc analysis of the INPULSIS data comparing 1061 IPF patients treated with antacids  at baseline versus 655 patients who did not receive antacids at baseline. This dataset did not show any beneficial effect of antacids on lung function as demonstrated by lack of effect on the change in FVC (n = 394) compared to the “antacid group” (n = 244). In other words, there were presumably more patients who were taking the antifibrotic drug nintedanib in the “no antacid group.” Thus, the beneficial effect of nintedanib is likely to influence the possible efficacy of antacids. In fairness, the data should have separated the placebo arm and the nintedanib arm and then compared the effect of antacid medications within the placebo arm and/or within the nintedanib arm.The curiosity of documented beneficial outcomes associated with the use of PPIs has led to querying the data gathered from the INPULSIS (nintedanib) , as welle in FVC . Howevern = 291) in comparison to the “no antacid therapy” group (n = 333). After adjustment for several confounders, this study showed positive trends favoring the antacid group (of whom about 90% were on PPIs) in terms of IPF-related mortality, death or 6-min walk distance (6MWD) decrease by 10% or more, progression-free survival and all-cause mortality  versus pirfenidone alone (n = 350) found that the use of antacids (of whom about 90% received PPIs) on top of pirfenidone was associated with statistically significant reduction in the loss of lung function by 10% or more (≥10% FVC decrease) compared to pirfenidone alone (p = 0.0273)  . Antacid 0.0273)  did not  0.0273) . Similar 0.0273)  has supe1 (NCT02085018). However, larger and longer duration studies are required to definitively address the value of PPIs in IPF. The conditional recommendation of PPIs for the treatment of IPF by the North American, European, Japanese and Latin American Thoracic Societies . Nonetheless, the widespread issue of negative clinical trials in IPF needs to shape the design and commencement of future clinical trials, as well as spark more innovative strategies to discover newer molecular targets and refine existing ones. In addition, there is a need to increase ancillary support for translational research aimed at performing high-throughput (HTS) and ultra-high-throughput (uHTS) drug screening of libraries composed of existing drugs and NCEs.So far, a number of molecules and signaling pathways have been targeted in IPF. Some of these targets include cytokines, kinase enzymes, collagen scaffold, telomerase, phosphodiesterase, endothelin receptors, integrins, oxidative stress, coagulation, angiogenesis, inflammation, and the renin-angiotensin system. Despite the array of targets, the failure rate is daunting , for therapeutic use of new indications including IPF.