Therefore, we generated mice that lack CaBP2 or CaBP1/caldendrin (–/–Cabp2 and –/–Cabp1) to test whether these CaBPs are essential for normal retinal function. By immunohistochemistry, the overall morphology of –/–Cabp1 and –/–Cabp2 retinas and the number of synaptic ribbons appear normal; transmission electron microscopy shows normal tethered ribbon synapses and synaptic vesicles as in wild-type retinas. However, whole-cell patch clamp recordings showed that light responses of retinal ganglion cells of –/–Cabp2 and Cabp1–/– mice differ in amplitude and kinetics from those of wild-type mice. We conclude that CaBP1/caldendrin and CaBP2 are not required for normal gross retinal and synapse morphology but are necessary for the proper transmission of light responses through the retina; like other CaBPs, CaBP1/caldendrin and CaBP2 likely act by modulating presynaptic Ca2+-dependent signaling mechanisms.Calcium-binding proteins (CaBPs) form a subfamily of calmodulin-like proteins that were cloned from the retina. CaBP4 and CaBP5 have been shown to be important for normal visual function. Although CaBP1/caldendrin and CaBP2 have been shown to modulate various targets  Ca2+-binding proteins (CaBPs), including the calmodulin-like CaBPs, exert Ca2+-dependent effects on specific target proteins—e.g., ion channels. To determine whether CaBP1/caldendrin and CaBP2 are important for normal retinal function, we used CaBP1/caldendrin- and CaBP2-deficient mice. Although these proteins are not required for retinal development and maintenance, CaBP1/caldendrin and CaBP2 are important for normal transfer of light signals through the retina.Electrical signals generated by the photoreceptors in response to incident light are processed by diverse retinal neurons before being sent to the brain. Ca2+-binding proteins with high homology to calmodulin  and long (L-) forms of these proteins . A thirdby CaBP2 .–/–Cabp1) and newly generated CaBP2 KO (–/–Cabp2) mice to investigate the importance of CaBP1/CD and CaBP2 for normal visual function. We find that both CaBP1/CD and CaBP2 regulate ganglion cell light responses and have different effects on their contrast sensitivity.Despite the identification of a large variety of interacting partners, the contributions of CaBP1/CD and CaBP2 to the transmission of light responses through the retina is unknown. In this study, we used CaBP1/CD knockout , Alexa Fluor 488 goat anti-rabbit , Alexa Fluor 555 goat anti-rat , Alexa Fluor 568 goat anti-mouse IgG2a , Alexa Fluor 488 goat anti-mouse IgG1 , anti-calretinin , anti-synaptotagmin 2 , anti-Ctbp2/ribeye , anti-NK3R , anti–green fluorescent protein , anti-calsenilin , and anti-CaBP1, anti-CaBP2, and anti-CaBP5 .m glycine buffer, pH 2.5, and dialyzed overnight against PBS.Anti-CaBP1/CD and anti-CaBP2 polyclonal antibodies were raised in rats by subcutaneous immunization with purified S-CaBP1 or S-CaBP2 recombinant proteins mixed with Freund’s adjuvant . For affinity purification of rat anti-CaBP1/CD or anti-CaBP2 antibodies, purified CaBP1 or CaBP2 was coupled to cyanogen bromide–activated Sepharose  according to the manufacturer’s protocol. After loading of a 10-fold dilution of the sera in PBS, the columns were washed with 20 volumes of PBS. The bound antibodies were eluted with 0.1 Cabp2 gene was amplified first from C57Bl/6 genomic DNA by PCR with primer FH939 (5′-GTCGACTAAGTAGCTGAGACCAGAAGAGATCGAAG-3′) that was extended with a SalI restriction site and includes a stop codon in all three open reading frames and primer FH940 (5′-GGTACCAGGAGGGCTCAGTTGCTCACATTA-3′) that was extended with a KpnI site. After sequencing of this 2.1-kb fragment, it was cloned into the targeting vector SalI and KpnI opened between the neomycin phosphotransferase gene and herpes simplex virus thymidine kinase gene. The long arm of ∼5.2 kb covering the promoter region of the Cabp2 gene upstream the ATG was amplified in two fragments. The upstream fragment of 2 kb was amplified by PCR with primer FH937 (5′-GCGGCCGCTCGTGGTTTCAGGTGCTCTACACA-3′) that was extended with a NotI site and primer FH947 (5′-TAAGGTCTTAGAGGGTCTGACAGG-3′) that covers a SpeI restriction site. A 3.2-kb fragment upstream of the CaBP2 initiation codon was amplified by PCR with primer FH938 (5′-ACCCAGGTTTCTGGCCTTATGTCT-3′) that also covers the SpeI restriction site and FH948 (5′-TACCGACTGACTCATGCCTAGGTT-3′) that hybridizes a few bases downstream of the CaBP2 initiation codon. All fragments were cloned in the pCRII-TOPO vector and sequenced. A tdTomato vector  was modified by mutagenesis using QuikChange Lightning Multi Site-Directed Mutagenesis  to introduce a NheI site after the SV40 polyadenylation site with primer FH1043 (5′-GTATCTTAAGGCGTAGCTAGCAAGCTTTAATATTTTGTTAAAATTCGC-3′) and delete the internal NcoI site in tdTomato with primer FH1044 (5′-CGTAATGCAGAAGAAGACGATGGGCTGGGAGGCCTCC-3′). tdTomato was then fused to the CaBP2 promoter as a fragment NcoI-NheI and transferred together in the targeting vector as NotI-BglII and BglII-NheI fragments.All animal procedures were performed in accordance with the University of Washington Animal Care and Use Committee’s regulations. A ∼2.1-kb fragment covering part of exon 2 to part of intron 5 of neo cassette and FH1139 (5′-TACACAGGCTCACCGAGACATCAT-3′) hybridizing approximately 163 bp downstream of the 3′ end of the short arm in the Cabp2 gene and amplifying a fragment of ∼2.3 kb. A control PCR for the wild-type (WT) gene was made with primers FH1139 and FH1140 (5′-ACCAGGCATGGAGTTGGGTATGAA-3′) hybridizing in intron 2A, 480 bp upstream of the 5′ end of the short arm of the Cabp2 gene and amplifying a fragment of ∼2.75 kb. Targeted disruption of the Cabp2 gene was then confirmed by Southern blot analysis. Ten micrograms of genomic DNA was digested with MfeI and hybridized with a 0.6-kb 5′-end probe located 100 bp upstream of the 5′ end of the long arm (Cabp2 gene is targeted.The KpnI linearized targeting vector was electroporated into B6/BLU embryonic stem cells. Recombinant clones were selected on medium containing G418. Transfected embryonic stem (ES) cell clones were first screened through PCR analysis. To screen for homologous recombination, we used primers FH 1064 (5′-GGGTCGTTTGTTCGGATCCTCTAGAGTC-3′) located in the long arm . This pragouti offspring were genotyped by PCR to verify germline transmission. Confirmation of Cabp2 gene targeting was first performed with primers FH1139, FH1140, and FH1064 as indicated above. For routine genotyping of the offspring, the WT allele was identified with primers FH1214  and FH1218  that generate a PCR product of 0.4 kb. The targeted Cabp2 allele was identified with primers FH1218 and FH1384  that produces a PCR product of ∼1.07 kb.One targeted ES clone was injected into C57BL/6J blastocysts. One 80% male chimera was crossed with C57BL/6J mice, and Total RNAs were isolated from mouse retina using RNeasy kit . Total RNA (1 μg) was subjected to first-strand cDNA synthesis using Superscript III reverse transcriptase and oligo(dT) in a volume of 20 μl according to the manufacturer’s protocol (Invitrogen). Short and long CaBP1 were amplified with common forward FH705 (5′-CACCATGGGCAACTGCGTCAAGTCG-3′) and reverse FH706 (5′-TCAGCGAGACATCATCCGGACAAAC-3′) primers. Caldendrin was amplified with primers FH1089 (5′-ACACCAATCATATCTGCCGTCTCC-3′) and FH1093 (5′-GCGATGGGGAGGAACGGGGGCT-3′). Short and long CaBP2 were amplified with common forward K122 (5′-TCCGGGCCTGGCATGGTTC-3′) and reverse FH1410 (5′-CCGAACAAATTCTTCAAAGTCAACC-3′). Amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) forward (5′-GAAGGGCTAATGACCACAGTCCAT-3′) and GAPDH reverse (5′-TAGCCATATTCGTTGTCGTACCAGG-3′) was used as a positive control. The PCR conditions were as follows: 94°C for 2 min, 35 cycles of 94°C for 15 s, 64°C for 30 s, and 72°C for 1 min, then 72°C for 7 min.–/–Cabp1, –/–Cabp2, –/–Cabp1/–/–Cabp2, or WT mice of either sex were fixed in 4% paraformaldehyde in 0.1 m phosphate buffer, pH 7.4 (PB), for 1 h. After fixation, tissues were immersed in a graded sucrose series to 20% sucrose in 0.1 m PB and embedded in 33% optimum cutting temperature compound  diluted with 20% sucrose in PB before being frozen. Eye tissues were cut in 12-µm sections. To block nonspecific labeling, retinal sections were incubated with 3% normal goat serum in PBST buffer  for 20 min at room temperature. Sections were then incubated overnight at 4˚C with purified rat anti-CaBP1/CD 1:50 dilution or rat anti-CaBP2 1:20 dilution. Alexa Fluor 555–conjugated goat anti-rat IgG and Hoechst stain (1:2000) were reacted with the sections for 1 h at room temperature. The sections were rinsed in PBST and mounted with Prolong antifade reagent (Invitrogen). As indicated, some sections were incubated overnight at 4°C with a mix of rat or rabbit anti-CaBP1/CD or anti-CaBP2 antibodies and rabbit anti-NK3R at 1:500 dilution, mouse anti-Syt2 at 1:200, mouse anti-calretinin at 1:1000, chicken anti-GFP at 1:1000, mouse anti-calsenilin at 1:1000, or rabbit anti-CaBP5 at 1:200. A mix of Alexa Fluor 555–conjugated goat anti-rat IgG (1:400) and Alexa 488–conjugated goat anti-rabbit IgG (1:400), Alexa Fluor 488–conjugated goat anti-mouse IgG (1:400), or Alexa Fluor 488–conjugated goat anti-chicken IgG (1:400) was then reacted with the sections for 1 h at room temperature.Eyecups from 6-week-old to 3-month-old For the analysis of whole-mount retinas, the mouse retinas were fixed for 1 h in 4% paraformaldehyde in PB before dissection, incubated with 5% normal goat serum in PBST buffer overnight at 4°C, and incubated overnight at 4°C with a mix of anti-Syt2 (1:200) or anti-NK3R (1:500) and anti-Ctbp2 (1:1000). After three washes for 15 min in PBST, a mix of Alexa Fluor 568 goat anti-mouse IgG2a (γ2a) and Alexa Fluor 488 goat anti-mouse IgG1 (γ1) or a mix of Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 555 goat anti-rabbit was reacted with the sections overnight at 4°C. After three washes in PBST, the retinal whole mounts were mounted with the photoreceptor side down in Prolong antifade reagent and analyzed under a confocal microscope . Immunofluorescent images were obtained with 63×/1.4, 40×/1.1, or 40×/1.3 NA objectives (Zeiss).–/–Cabp1, and –/–Cabp2 retinas were acquired using a 63×/1.4 objective at a voxel size of 0.028 × 0.028 × 0.359 µm (x-y-z). The sublamina containing the axon terminals of cone OFF bipolar type 1 and type 2 was identified by colabeling with anti-NK3R and anti-Ctbp2. The axon terminals of cone ON bipolar type 6 were visualized by colabeling with anti-syt2 and Ctbp2. Ctbp2-labeled synaptic ribbons were counted in images of six randomly chosen 25-μm2 areas across the retina of four mice for each phenotype. The statistical analysis was done using the two-tailed Student’s t-test. Data are expressed as mean ± SE.For quantification of the Ctbp2-labeled synaptic ribbons, confocal images of en face views in whole mounts of WT, –/–Cabp1, and –/–Cabp2 mice of either sex were fixed in 4% glutaraldehyde in 0.1 m sodium cacodylate buffer at 4°C. After four washes in 0.13 m PB, eyecups were dissected and postfixed in 1% buffered osmium tetroxide for 2 h on ice. After another four washes in PB, the retinas were en bloc stained in 1% uranyl acetate overnight at 4°C, dehydrated through a graded series of ethanol and two incubations in propylene oxide, and infiltrated in a 1:1 mixture of propylene oxide:epon araldite and two incubations in epon araldite, before polymerization at 60°C. Seventy-nanometer sections were cut on a Leica UC7 ultra microtome and stained with Reynolds lead citrate. The sections were viewed and images recorded on a JEOL 1230 electron microscope . Images of bipolar cell terminals were analyzed in the outer part of the inner plexiform layer within 2–5 μm from the inner nuclear layer where type 1 and type 2 cone OFF bipolar cell axon terminals are located. Images of photoreceptor synapses with bipolar cells were taken in the outer plexiform layer.Eye cups from WT, –/–Cabp1, or –/–Cabp2 mice (2/5% CO2) Ames medium  at ∼32–34°C. Isolated retinas were flattened onto poly-l-lysine slides : 105 Cs methanesulfonate, 10 tetraethylammonium chloride, 20 HEPES, 10 EGTA, 2 QX-314, 5 Mg-ATP, 0.5 Tris-GTP . Alexa 594 dye (100-200 microM) was included in the intracellular solution to image the ganglion cells post recording as shown in t-test; *p < 0.05, **p < 0.01, ***p < 0.001; error bars indicate SD.Experiments were conducted on whole-mount retinal preparations taken from dark-adapted WT, –/– mice . Isolate–/–Cabp2 mice, we designed a targeting vector to replace exon 1 and exon 2a of the Cabp2 gene with tdTomato and a PGK-neo cassette as shown in Cabp2 allele was further verified using Southern blot -PCR using retinal RNA of –/–Cabp1 mice , and OFF sustained (OFF S). Extensive work on these alpha-like ganglion cells has identified type-specific response properties generated from distinct circuit mechanisms . Recordi–/–Cabp2 retinas ; this suggests that alterations in the transmission of the light signal likely occur downstream of the bipolar cell dendrites.We investigated the light responses of alpha ganglion cells to determine the importance of CaBP1/CD and CaBP2 for proper transmission of light responses across the retina. Compared with responses of ganglion cells from WT retina, we observed a ∼50% increase in the amplitude of the excitatory postsynaptic currents of OFF T ganglion cells of –/–Cabp1 mice. The opposite changes in response range could be explained if the bipolar cells that provide input to OFF S and OFF T ganglion cells express different targets for CaBP1/CD. Alternatively, CaBP1 may differently shape the synaptic input to these two types of OFF ganglions cells.Interestingly, the two OFF alpha ganglion cell types displayed similar changes in kinetics but opposite changes in response range in The absence of CaBP2 resulted in a decrease in the response range of the ON alpha ganglion cell response. The absence of CaBP1/CD slowed response kinetics but did not change the response range of the excitatory synaptic currents of the ON alpha ganglion cells. Because CaBP1 is not found in cone ON bipolar cells, the effect of CaBP1 deficiency on ON alpha ganglion cell excitatory inputs could result from alterations in responses of amacrine cells that provide presynaptic inhibition on type 6 bipolar cells and shape the kinetics of excitatory inputs of ON alpha ganglion cells. The complex interplay between ON and OFF pathways means that other explanations are possible as well.2+ channels  and Cav3  complex. We found here that deficiency of CaBP1/CD also results in slower response kinetics than in WT animals. If CaBP1/CD were to have similar effects on synaptic vesicles priming, it could affect the kinetics of the light responses.CaBPs have also been shown to interact with other proteins playing an important role in neurotransmitter release. For example, CaBP5 interacts with Munc18-1, a component of the synaptic vesicle cycle. CaBP5 was suggested to have a role in synaptic vesicle priming by binding to a specific domain of Munc18-1 necessary for its interaction with the soluble –/–Cabp1 and –/–Cabp2 mice gives us a first insight into the important role of CaBP1/CD and CaBP2 for normal transmission of the light responses through retinal circuits. Although the effects of CaBP1/CD and CaBP2 deficiency might result from a change of modulation of voltage-gated calcium channels, other cellular processes might also be altered. Future experiments investigating the interacting partners for CaBP1/CD and CaBP2 in bipolar cells will help to explain the visual phenotypes in the CaBP KO mice and the cellular functions of these proteins in the retina.In conclusion, the phenotype of


Previous studies on hybrid dielectric-graphene metasurfaces have been used to implement induced transparency devices, while exhibiting high Q-factors based on trapped magnetic resonances. Typically, the transparency windows are single wavelength and less appropriate for polarization conversion structures. In this work, a quarter-wave plate based on a hybrid silicon-graphene metasurface with controllable birefringence is numerically designed. The phenomena of trapped magnetic mode resonance and high Q-factors are modulated by inserting graphene between silicon and silica. This results in a broader transmission wavelength in comparison to the all-dielectric structure without graphene. The birefringence tunability is based on the dimensions of silicon and the Fermi energy of graphene. Consequently, a linear-to-circular polarization conversion is achieved at a high degree of 96%, in the near-infrared. Moreover, the polarization state of the scattered light is switchable between right and left hand circular polarizations, based on an external gate biasing voltage. Unlike in plasmonic metasurfaces, these achievements demonstrate an efficient structure that is free from radiative and ohmic losses. Furthermore, the ultrathin thickness and the compactness of the structure are demonstrated as key components in realizing integrable and CMOS compatible photonic sensors. With proper dimensions of the antenna, a 90° phase difference is obtained as shown in Fig. Txx = Tyy occurs near the resonance, defining an ideal QWP condition. Additionally, by sweeping through different values of the length L2 while keeping L1 fixed (L1 = 440 nm), the resonance amplitudes associated with different electric and magnetic modes can be varied. An acceptable phase bandwidth range within ± 10° was obtained when L2 = 365 nm for RCP, and L2 = 450 nm for LCP, as shown in Fig. λ = 1.48 μm, the undoped graphene (EF = 0 eV) causes high penetration of electric fields into the silicon dipoles and a large phase difference between the x and y components of scattered light (≈150°) occurs. However, as EF approaches 0.8 eV, the in-plane properties (εx = εy) increase the surface conductivity of graphene, resulting in a reduced penetration into the silicon and a ΔΦ≈90° at λ = 1.49 μm.The graphene effect is beneficial for tuning the phase components and the phase retardation of the transmitted electric fields. Firstly, the components of the incident LP light are decomposed into the orthogonal arms of the silicon antenna. Each dipole resonance imprints a different phase pattern on the scattered light. Specifically, near the resonance, each dipole resonance shifts the phase of the incident electric field in the range [− xed L1 = 40 nm, thL1 = 450 nm, L2 = 370 nm, and W = 60 nm are shown in Fig. λ = 1.5 μm, the Stokes parameter ratio |S3/S0|≈ ± 1, where a + 1 value indicates a perfect RCP and a − 1 indicates a perfect LCP output. Here, S0 = |Ex|2+|Ey|2 and S3 = 2ExEy sinΔΦ are the Stokes parameters. The degree of transmission intensity is determined by S0, i.e., a value > 50% is acceptable. Figure The calculated Stokes parameters and polarization ellipse dimensions for the hybrid structure with Tyx and Txx are cross and co-polarization terms, respectively. Within the wavelength range λ = 1.48 μm and λ = 1.51 μm, the efficiency is ≈96% for RCP and ≈90% for LCP outputs. However, at λ = 1.52 μm, the efficiency slightly drops to ≈80% for LCP. As shown in Fig. Ex/Ey≈1 and phase shift ΔΦ≈90° are obtained in a wide range. When α = 48°, an accurate QWP condition is obtainedwhere Pi at the wavelength λ = 1.49 μm. In Fig. ΔΦ≈90°) occurs in the region where the transmittance is above 80%, as shown Fig. Additionally, the transmission phase profile defining the form birefringence was calculated as a function of the periodicities y-planes of the silicon/graphene structure was designed as shown in Fig. νF, and switches the direction of flow of electrons. Additionally, the bias voltage changes the carrier density of graphene which in turn leads to a change in its electrical conductivity and permittivity. In this configuration, the structure forms a quasi-parallel plate capacitor model with an electrostatic capacitance per unit area, C, defined as C = εsiε0/Px, where εsi is the dielectric permittivity of silicon. The Fermi energy, ns) and the electrostatic capacitance per unit area (C) scale the Fermi energy through the gate voltage, that is, ns = CVG/e. Consequently, an increment in Px decreases both the carrier concentration in graphene and the capacitance per unit area. As a result, as shown in Fig. Application of gate voltage bias across the λ = 1.5 μm, the two states of circular polarization can be encoded as two binary states, 0 and 1. The logic state 0 corresponds to the reverse voltage − 47.5V while the logic state 1 corresponds to the forward voltage 47.5V, as shown in Fig. ΔΦ≈0°, can be observed when the gate voltage is at − 25 V . This observation shows a non-linear response in the phase change at − 47.5, − 25, and 47.5 V, attributed to a variation in capacitive coupling as graphene becomes more conductive because of a change in the carrier density and gate voltage. In comparison to other wavelengths in the near-infrared, 1.5 μm shows the optimum point for switching the circular polarization states of the scattered light.At S3 illustrates the degree of circular polarization as a result of the gate voltage biasing. The − 1 and 1 limits denote the ideal polarization conversions from a linear state to LCP and RCP states, respectively. Between the wavelengths λ = 1.49 μm and λ = 1.52 μm, the degree of circular polarization approaches unity (> 90%) for both states, confirming the most appropriate operation region of the structure as a QWP.In Fig. z component of the electric field, calculated at the design wavelength λ = 1.5 μm at z = 0. The distribution shifts as the voltage is reversed from 47.5 to − 47.5 V. The change in electrical conductivity and carrier density of graphene results in a rotation of the trapped magnetic mode around the silicon structure.Figure %) in the near-infrared (λ = 1.45 to 1.54 μm). Unlike in plasmonic metasurfaces, these achievements demonstrate high efficiency devoid of radiative and ohmic losses. Additionally, the structures are compact and have an ultrathin thickness, appropriate for compatibility and integration with CMOS and photonic devices. Meanwhile, graphene is feasible and can be grown using chemical vapor deposition on the substrate while the silicon structure can be fabricated using standard lithographic methods.In summary, birefringence controllability of a hybrid silicon/graphene metasurface polarization converter has been numerically designed. Trapped magnetic modes and high Q-factors are modulated by integrating graphene and silicon. Two configurations of the hybrid structure have been shown, one with a gate voltage bias and the other without. In the voltage-biased structure, birefringence performance is shown through reversal of the gate voltage. From an incident LP light, a reverse bias voltage (- 47.5 V) produces an RCP output and a forward bias voltage (47.5 V) produces an LCP output. Hence, a dynamic switching performance is achieved. For the free-space configuration, QWP performance is shown through manipulation of the dimensions of silicon and the Fermi level of graphene. In both designs, a more stable and broader bandwidth is obtained than in structures without graphene. The designs show higher degrees of polarization conversions (>96


The development of humidity sensors with simple transduction principles attracts considerable interest by both scientific researchers and industrial companies. Capacitive humidity sensors, based on polyimide sensing material with different thickness and surface morphologies, are prepared. The surface morphology of the sensing layer is varied from flat to rough and then to nanostructure called nanograss by using an oxygen plasma etch process. The relative humidity (RH) sensor selectively responds to the presence of water vapor by a capacitance change. The interaction between polyimide and water molecules is studied by FTIR spectroscopy. The complete characterization of the prepared capacitive humidity sensor performance is realized using a gas mixing setup and an evaluation kit. A linear correlation is found between the measured capacitance and the RH level in the range of 5 to 85%. The morphology of the humidity sensing layer is revealed as an important parameter influencing the sensor performance. It is proved that a nanograss-like structure is the most effective for detecting RH, due to its rapid response and recovery times, which are comparable to or even better than the ones of commercial polymer-based sensors. This work demonstrates the readiness of the developed RH sensor technology for industrialization. Humidity is defined as the water vapor amount in air. It is the most abundant greenhouse gas and an important abiotic factor influencing the human life. To describe humidity in a precise way, several items are commonly used, which are absolute humidity (AH), relative humidity (RH), specific humidity (SH), and dew point . The RH Recently, the state of the art of humidity monitoring has evolved rapidly and continuously. However, the wide application range of devices being able to record humidity is behind the intensive scientific studies dedicated to this area. Their most important fields of use are indoor application, plants growth in agricultural market, and control of industrial processes, electronics, and semiconductor area, as well as medical processes ,5,6,7,8.A survey of the literature leads to a classification of humidity sensors based on their working principle as capacitive, electrical conductivity (or resistive) and optical sensors. It is obvious that, except optical sensors, a humidity sensor is defined as a combination of a transducer and a sensing material to humidity.Interdigitated transducers (IDT), also known as interdigital electrodes, have been intensively studied and used for unlimited applications due to the possibility of measuring both capacitance and resistance ,10. An I2O3, TiO2, SiO2, and spinel compounds with AB2O4 structural composition, are known to change their electrical properties, such as resistance, in the presence of humidity [Ceramic materials, like Alhumidity . The firhumidity . Each sihumidity , thus a humidity . This mehumidity . Decreas2, In2O3 [Semiconducting materials, like SnO2, In2O3 , and per2, In2O3 , are pro2, In2O3 . This le2, In2O3 .The third type of humidity-sensing material is an organic material being commonly a polymer. It is important to underline that polymers are an emerging candidate in many sensing applications due to low cost, commercial availability, and easy deposition on different transducers. Besides this, a photosensitive polymer adds advantages related to rapid polymerization, elimination of volatile organic solvents usage, and reduction of technological process steps. A polymeric layer in contact with water vapor shows a change in its conductivity or its dielectric constant ε. As water diffuses into a hydrophobic polymer layer like polyimide, poly(methyl methacrylate) (PMMA), cellulose acetate butyrate (CAB), or poly (PETT), the matrix absorbs water and only its relative permittivity ε undergoes changes . SeveralNowadays, capacitive humidity sensors dominate the market. Polymeric layers as a sensitive layer in combination with interdigitated electrodes are intensively studied . DespiteA chemical sensor is a device that transforms chemical information, i.e., the analyte concentration, into an electrically useful signal. Basically, it consists of a transducer and a sensitive layer. The chemical information is obtained either due to a chemical reaction or due to a sorption process between the analyte and the sensitive layer. In our study, we fabricated capacitive humidity sensors, which monitor any changes of in the dielectric properties of the appropriate sensing material. The transducer was a pair of planar interdigitated electrodes. The sensing material was polymer-based material called polyimide.2. Finger width, length, and pitch were set to 6 µm, 2 mm, and 12 μm, resulting in 83 and 42 fingers, respectively.A pair of interdigitated microelectrodes was produced by standard photolithography on a 620 µm thick glass substrate. For operating at elevated temperatures, quartz substrates are the best choice. After cleaning the glass substrate wafer, a thin adhesion layer of TiW (40 nm) followed by a thick gold layer (140 nm) were deposited by sputtering deposition technique. The electrodes were patterned by photolithography technique using the desired mask. The pattern included two wires around the interdigitated electrodes, one to heat the sensor and the other to measure the surrounding temperature . Each ch2 inert atmosphere. The curing temperature was 400 °C.The polyimide used for humidity sensing layers was prepared from a photosensitive solvent developable polyamide acid ester. First, the substrate surface was cleaned with a stripper in an ultrasonic bath from any contaminants and organic impurities prior to the coating process in order to ensure a good adhesion of the sensing layer to the glass and metallic electrodes surface. The polyimide material was spin coated homogeneously on top of the planar electrodes on the 6-inch glass wafer. Different layer thicknesses were obtained, ranging from 1 µm to 11 µm. The spin coating parameters (spin speed and time) varied according to the desired final film thickness and the precursor initial viscosity. After spin coating, the films underwent a soft bake for a short period of time to drive out residual solvent. Then, the photosensitive layer was exposed to an appropriate dose using a mask for pad opening. The exposure dose was chosen based on the thickest layer in case of a variation of the coating thickness on the substrate. The wet development process was realized at room temperature for an optimal time and followed by flood exposure, without a mask, applied after developing to allow a complete crosslinking reaction. The imidization reaction took place between the amino group and the ester group resulting in an imine group. After completing the photolithographic process, the film was cured in a Yes Oven under N2 large individual chips. Each chip was mounted on an adapted printed circuit board (PCB), glued, and then gold wire bonded thereto. The PCB was equipped with zero insertion force (ZIF) connectors. The coated wafer with polyimide sensing material was diced into 4.7 × 3.9 mm2 or synthetic air.A reliable gas mixing apparatus, which is a part of a gas measurement setup, is an important system for characterizing gas sensors under desired gas mixtures. The dynamic volumetric method, described in reference , was adoUp to 10 chips could be placed into a cylindrical stainless steel chamber (sensor chamber), which is connected to the gas mixing apparatus a. ConseqThe electrical properties of the sensors were monitored by impedance spectroscopy using a Solartron gain-phase frequency analyzer model 1260 A controlled by a PC, permitting automated data collection. The impedance measurements could be carried out at different temperatures (from RT to 200 °C) by heating up the chip using the integrated heater around the electrodes. The measurements were performed from 1 MHz to 10 Hz to get the optimal frequency at which the sensor capacitance change linearly as a function of RH. Within another project, it is planned to develop a miniaturized system using the AD chip. The AC Voltage excitation of the AD chip is around 38 kHz. For this reason, most of the experimental measurements are done at 40 kHz.For this study, the capacitance is recorded at 25 °C under different frequencies . The senthV: threshold voltage (1.5 V)ddV: positive supply voltage (3.3 V)REFC: reference capacitorN: number of charge transfer cycles needed to charge the sC.An evaluation kit with high speed data acquisition was designed. Additional features are low power, low cost, compact size, and network compatibility. The used microcontroller STM32-L476RG possesses a low-power design, several Analog-to-Digital converters (ADC), and a touch sensing controller for capacitive measurements via the charge transfer method on board. The touch sensing controller consists of a number of internal switches. After loading the sensor capacitor with a constant voltage, the charge is transferred by the switches to an external reference capacitor (104 times bigger). This process is repeated N times, until a threshold voltage is reached on the reference capacitor. The capacitance of the sensor is then calculated by (1)Cs=−CAn extension circuit board was developed, which holds sensor connectors, heater driver circuit, resistance temperature detectors (RTD), and reference capacitors for the charge transfer method. Additionally, PT1000 and SHT25 were added as reference sensors and connectors for communication protocols (UART and I2C). The evaluation kit can simultaneously measure three sensors by measuring the capacitance and controlling the operating temperature by a proportional-integral-derivative (PID) algorithm. It permitted measuring the response and recovery times of the developed sensors by ensuring a short measurement interval and quick gas exchange ability. In order to use the evaluation kit together with the gas mixing apparatus, a PEEK lid was designed and screwed with a sealing ring onto the PCB, where three sensors sockets were positioned b. The sa2) were coated with a polyimide layer and examined under IR radiation at different humidity levels by a FTIR spectrometer. A background reference curve is measured with an uncoated sample from the same silicon wafer. A first measurement is performed with a polyimide coated sample under dry condition inside the FTIR interior space at nearly 0% RH  is an extremely reliable method that provides chemical bond and molecular composition present of any partially transparent material. To realize FTIR spectroscopy for our purpose, silicon test wafer substrates  is a simulation software, which applies finite element simulation to a 3D model of the structure to be studied in this work. The CAD is used to build a 3D model that consists of several layers: glass substrate, gold layer, sensitive layer , and air layer. The derived capacitance of a bare IDT by FEM simulations agree well with the one measured by the impedance analyzer. By combining simulations with different dielectric constants of the sensitive layer and the measured sensitivity of the coated IDT, the correlation between the dielectric constant of the polyimide layer and the relative humidity was determined. Therefore, at 25% RH the dielectric constant of polyimide is equal to 3.5.PI~3.5). The values of the penetration depth are obtained from COMSOL plots of the electric field. Calculation of the penetration depth is simulated to determine the maximum reasonable polyimide layer thickness. In the last years, high aspect ratio structures from nature have inspired engineering and scientists to develop new materials. The nanostructures, having superhydrophobic surface properties, is still attracting considerable attention for application in photovoltaic , biosensIn the case of polyimide material, Lee et al. proposed that etching of the layer at pressure equal to 100 mTorr for 5 min results in a nanograss structure . A pressIn the present work, the plasma etching process of polyimide films is carried out under oxygen plasma at two different pressures, 50 mTorr and 720 mTorr, using a reactive ion etching chamber from Plasmalab. Oxygen plasma is generated by a radio frequency (RF) power source. The RF power for both working pressures is fixed to 113 W. The bias voltage, which built up between oxygen source and sample chuck, is 380 V and 140 V for lower and higher etching pressure, respectively. The bias voltage reflects the kinetic energy of the etching reactants. Hence, the oxygen ions with the lower pressure hit the polyimide surface with a higher kinetic energy. The etching rate of polyimide layer is determined by measuring the layer thickness by a profilometer after an etching time of 2 min. The plasma etched samples are analyzed by SEM, as presented in Previous theoretical work established a correlation between pressure and etching rate  to explaThe realized etching process under oxygen plasma at low pressures, 50 mTorr, yields nanograss-like polyimide structures with higher etching rate . In thisA series of photosensitive polyimide layers are deposited onto IDTs. The layers present different thickness and surface morphologies. To characterize the performance of chemical sensors under development, sensors are placed into a hermitic test measurement chamber and the sensors capacitance is monitored under different humidity levels by an impedance analyzer. A commercial humidity sensor, SHT25 device with a standard calibration, is selected for reference.RH=85% and CRH=6% are the measured capacitance at high and low level of relative humidity, respectively.The sensitivity of the sensors decreases from 15 to 9.5 fF/% RH by decreasing the polyimide thickness from 4.6 to 1.5 µm with a flat morphology. As described in Although a gas measurement setup, combining the gas mixing apparatus and electrical measurements using impedance analyzer, is a standard method to characterize the performance of chemical sensors under development, the response and recovery times, typically less than 3 min, are difficult to be accurately monitored for RH sensors under test. The reason is that some minutes (5 min) are needed to ensure a complete gas exchange inside the testing chamber . To overThe response and recovery times in this study are defined as the times required for the sensor capacitance to reach 90% of the maximum measured signal, when the humidity is changed between two humidity levels (here from 6% RH to 67%). As the sensing mechanism is based on diffusion, the thinner sensing layer shows a smaller response as well as a smaller recovery time. However, the nanograss-like layer shows the best performance even in comparison to commercial humidity sensors. An optimization of the transducer design and its miniaturization will lead to a further improved sensor performance. The measured capacitance, normalized, of the polyimide-based RH sensor (for both thicknesses and morphologies) as a function of time is shown in The RH level in the test chamber is varied from dry to 65% and back to the initial level 6% after some seconds. The sensor samples with a polyimide thickness of 4.6 µm having a flat morphology and 1.5 µm rough surface and 1.5 µm nanograss-like surface present the same response and recovery times once measured in the chamber of gas mixing setup. As mentioned in 2 or synthetic air.It is worth noting that the sensor responses are uncorrelated to the used ambient air. No significant differences are detected of measuring the sensors response under carrier gases NGenerally, the adsorption and desorption of a specific analyte carried by an inert gas into a sensing material matrix is governed by either diffusion process or thermodynamically controlled process or even both processes. In case of an ideal diffusion-controlled process, the response time and the recovery time should be equal. In the thermodynamically controlled process, the adsorption is an exothermic process, whereas desorption is an endothermic process. Consequently, desorption requires higher external energy which is reflected by a higher recovery time. An adsorbed molecule on the surface of a sensing layer at certain temperature remains stable at this state. A moderate increase of the sensor temperature, in the absence of any interaction between the sensing material and the analyte, leads to desorption of the desorbed molecule from the surface and returns into its initial gas form.2 plasma etch will be subject of future studies.As confirmed from the FTIR measurements, the polyimide material presents no chemical interaction between the polymer backbone and water molecules. However, the polyimide layer with rough and nanograss-like morphologies show a large difference between their response and recovery times (72%) to humidity. This large difference between the two parameters describing the humidity sensor is probably due to structure changes into the polyimide matrix induced by the plasma process. These structure changes in the polymer induced by the O2 plasma etch parameters. The nanograss morphology exhibits the best performance among the studied morphology types. The ideal polyimide layer thickness turns out to be equal to the penetration depth of the used interdigitated transducer. The sensor signal, capacitance, follows a linear dependence with RH. Polyimide sensing layers with nanograss morphology ensure the best humidity sensor performance regarding the response and recovery times. The optimal polyimide layer thickness and morphology can be chosen as a function of the envisaged application of relative humidity sensors and the used interdigitated transducer.In summary, a systematic examination of the sensor performance of a series of polyimide layers with different thickness and morphologies  exposed to different humidity levels are measured simultaneously in the same test chambers. We have successfully demonstrated that the nanostructure of polyimide to obtain nanograss can be well controlled by adjustment of a series of O


PD-L1/JAK2/PD-L2 co-amplification using fluorescent in-situ hybridization assay (FISH). PD-L1 positivity  was present in 32/77 (42%) and 33/71 cases (46%) using SP142 and SP263 antibodies, respectively. Concordance between the two anti-PD-L1 clones was high with only three (4%) discrepant cases. The strongest and consistent (10/11 cases) expression was observed in cHL and primary mediastinal B-cell lymphomas (3/3). Diffuse large B-cell lymphomas (DLBCL) were frequently positive (13/26) irrespective of subtype. Follicular (1/8), peripheral T-cell (3/11) and mantle cell (1/8) lymphomas were rarely positive, while small lymphocytic lymphoma/CLL and marginal zone lymphomas were consistently negative (3/3). Co-amplification/CNVs of PD-L1/JAK2/PD-L2 were observed in 3 cases of DLBCL and cHL, respectively. Of note, all three cHL-amplified cases were positive by FISH, but not by NGS. Since only a fraction of the IHC positive lymphoma cases were positive by FISH and NGS assays, other mechanisms are involved in PD-L1 upregulation, especially in DLBCL. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL.Targeted immunotherapy based on PD-1/PD-L1 suppression has revolutionized the treatment of various solid tumors. A remarkable improvement has also been observed in the treatment of patients with refractory/relapsing classical Hodgkin lymphoma (cHL). We investigated PD-L1 status in a variety of treatment resistant lymphomas. Tumor samples from 78 patients with therapy resistant lymphomas were immunohistochemically (IHC) investigated for the expression of PD-L1 using two antibody clones . Thirteen PD-L1+ cases were further analyzed for gene copy number variations (CNV) by NGS and for  PDCD1 gene) and one of its two known ligands, the programmed death ligand-1  are among the therapeutically most important checkpoint proteins that mediate tumor-induced immune suppression through T-cell downregulation , after removing outliers. Underperforming regions were excluded to ensure that the reportable regions were created from high quality data. A region was considered amplified if the normalized region depth was greater than or equal to 6 copies above the background ploidy. In order for a gene to be called amplified, a predefined proportion (90%) of the regions within that gene should be called amplified. The final output file included gene-level calls and region-level calls for each case.Batch sequencing run QC includes Cluster Density (supported number of clusters created per mm2 in appropriate range ~150–300); Reads Passing filter (Number of reads included in the data production) >65%; Q30 score—99.9% of base calls are accurate . Also the batch controls  must pass and produce acceptable and expected results. From a sample to sample passing quality metric, each sample must have >400 average depth of coverage overall or the case is repeated or reported out as indeterminate.The χ2 test was used for comparisons of the variables and groups while the Spearman correlation rank was applied for the correlation between the variables. The differences were considered significant at a p-value <0.05. All data were analyzed using IBM SPSS v.19 .The study included samples from 78 patients  diagnosed with refractory and/or relapsed lymphomas of B- and T-cell lineages. The mean patient’s age was 55.6 years . Histotypes included 11 cases of cHL , 26 cases of DLBCL , 9 cases of mantle cell lymphomas, 8 cases of follicular [FL]; 4 cases of marginal zone lymphoma; three cases of primary mediastinal B-cell lymphoma and small lymphocytic/chronic lymphocytic leukemia [SLL/CLL], respectively; single cases of lymphomatoid granulomatosis [LG], mixed FL/DLBCL, and B-cell lymphoma (not specified). The study also included 11 peripheral T-cell lymphomas .Results of PD-L1 expression were available for 77 cases while 71 cases had interpretable results for both anti-PD-L1 clones  was 32/77 (42%) by SP142 and 33/72 (46%) by SP263 antibody . The higThe mean percentage of PD-L1 positivity in tumor cells was 21% . Seven out of 10 positive cHL (70%) had PD-L1 positivity in 100% of the neoplastic cells Figs  and 2B aWe also evaluated PD-L1 positivity in adjacent inflammatory (reactive) cell population; Fifty-five out of 58 cases (95%) had PD-L1 positivity in reactive/inflammatory cells  in comparison with NHL positive cases . We found an excellent concordance between SP142 and SP263 clones  with onlResults of NGS/FISH assays are summarized in PD-L1 and PD-L2 without JAK2 gene alterations while another DLBCL case had co-amplification of all three genes. Among the 8 negative cases, four cases were cHL. These cases were further tested by FISH and three of them (75%) exhibited co-amplification of PD-L1/JAK2/PD-L2 genes (PD-L1 gene) while the third case harbored ≥6 copies of PD-L1 per tumor cell. One of the positive DLBCL cases was also confirmed by FISH while another, non-amplified DLBCL case by NGS exhibited no 9p24.1 alterations by FISH.Six out of 13 tested cases (46%) were positive by NGS or FISH . Two out of 10 tested cases by NGS exhibited co-amplification at 9p24.1: One case (DLBCL) harbored co-amplification of L2 genes ; two casImmune check point blockade with PD-1/PD-L1 has dramatically changed the cancer treatment paradigm with impressive results in several solid tumors as well as in cHL. PD-L1 overexpression has also been described in cancer subtypes beyond those that have been so far considered for immune check point inhibitors , 24–26, Patients with relapsed/progressive malignant lymphomas have limited therapeutic modalities and new therapeutic approaches are of crucial importance. Our study revealed over-expression of PD-L1 in cHL, DLBCL , a subset of peripheral T-cell lymphomas and a single case lymphomatoid granulomatosis. These findings are consistent with comprehensive surveys of Menter et al. [clones E1L3N  and SP142   and ChenPD-L1 gene [JAK2 (Janus kinase 2) gene involved in JAK2-STAT signaling that further may activate PD-L1 [PD-L2 gene, which is another ligand of PD-1. Similar to previous data, we showed that a subset of PD-L1+ lymphomas (both cHL and non-Hodgkin lymphoma) may harbor genetic alterations at 9p24.1 amplicon, which implies that the remaining non-amplified cases may have “adaptive” PD-L1 overexpression without underlying PD-L1 gene alterations [PD-L1 and PD-L2 without JAK2 gene alterations, which is in line with a recent study of Budczies et al. [PD-L1 copy number variations  across different cancer subtypes with direct impact on its protein and mRNA expression. In particular, cHL are characterized by consistent PD-L1/PD-L2 gene alterations [PD-L1 gene alterations in cHL. FISH method appears to be a more suitable methodology than NGS for assessing 9p24 status in tumors with a low cancer cell density as it allows for morphologic  identification of single amplified RS cells within heterogeneous (reactive) cell populations in cHL.There is increasing number of studies indicating that alterations at 9p24.1 affect PD-1 ligands (“9p24.1 amplicon”), particularly -L1 gene . This amte PD-L1  and PD-Lerations . One cass et al. . The auterations . One of PD-L1, PD-L1 and JAK2 genes. FISH assay may be more suitable than NGS assay for determination of PD-L1 alterations in cHL.We conclude that a substantial proportion of relapsed/refractory B-cell and T-cell non-Hodgkin lymphomas and nearly all classic Hodgkin lymphomas overexpress PD-L1 protein, implying a potential utility of checkpoint blockade therapy in these difficult to treat diseases. Anti-PD-L1 clones SP142 and SP263 exhibit an excellent concordance and both antibodies may be used for IHC detection of PD-L1 in tumors. We also confirm that a subset of refractory, PD-L1 positive lymphomas may harbor genetic alterations of 9p21.4 amplicon affecting S1 File(XLSX)Click here for additional data file.


Dystroglycan (DG) is an adhesion receptor complex composed of two non-covalently associated subunits, transcribed from a single gene. The extracellular α-DG is highly and heterogeneously glycosylated and binds with high affinity to laminins, and the transmembrane β-DG binds intracellular dystrophin. Multiple cellular functions have been proposed for DG, notwithstanding that its role in skeletal muscle appears central as demonstrated by both primary and secondary severe muscular dystrophic phenotypes collectively known as dystroglycanopathies. We recently analysed the molecular phylogeny of the DG core protein and identified the α/β interface, transmembrane and cytoplasmic domains of β-DG as the most conserved region. It was also identified that the IG2_MAT_NU region has been independently duplicated in multiple lineages.To understand the evolution of dystroglycan in more depth, we investigated dystroglycan gene structure in 35 species representative of the phyla in which dystroglycan has been identified . The gene structure of three exons and two introns is remarkably conserved. However, additional lineage-specific introns were identified, which interrupt the coding sequence at distinct points, were identified in multiple metazoan groups, most prominently in ecdysozoans.A coding DNA sequence (CDS) intron that interrupts the encoding of the IG1 domain is universally conserved and this intron is longer in gnathostomes (jawed vertebrates) than in other metazoans. Lineage-specific gain of additional introns has occurred notably in ecdysozoans, where multiple introns interrupt the large 3′ exon. More limited intron gain has also occurred in placozoa, cnidarians, urochordates and the DG paralogues of lamprey and teleost fish.The online version of this article (doi:10.1186/s13104-016-2322-x) contains supplementary material, which is available to authorized users. Drosophila melanogaster and Caenorhabditis elegans. It forms a bridging element that connects the internal cytoskeleton to basement membrane extracellular matrix [Dystroglycan (DG) is an adhesion receptor complex that provides mechanical stability to a wide variety of cells and tissues in mammals, zebrafish, r matrix . In thisr matrix . β-DG spr matrix .DG has been related to the function of skeletal muscle since its initial identification in rabbit sarcolemma . The calLeishmania mexicana (PDB:2C34) (Z-score of 5.1 and an RMSD of 3.2 Å over 82 residues) [The two subunits of DG are transcribed from a single gene. The domain organization of the primary protein product is as follows: a signal peptide; immunoglobulin-like domain 1 (IG1); S6 domain  .Fig. 1Arcis) of carbohydrate moieties that protrude from the neighbouring mucin-like region, and therefore could have additional functions within the glycosylation and maturation pathway of the dystroglycan precursor molecule [For the maturation of α-DG, the N-terminal region (IG1 and S6 domains) is considered highly important. Indeed, the N-terminal region in isolation displays a residual laminin-binding activity  and is lmolecule .Mnemiopsis leidyi and Pleurobrachia bachei [Nematostella vectensis (6X).Recently, we conducted the first extensive evolutionary study of the dystroglycan core protein and demonstrated a high degree of conservation in all metazoan phyla except ctenophores, where DG is absent from the two available species, a bachei . Our stua bachei . A majorDrosophila melanogaster [Apart from information on the DG gene in a few mammalian species , 23 or onogaster , no detanogaster . EspeciaExaiptasia pallida  intron (mini-intron) that interrupts the encoding of the S6 domain of α-DG  also has two DG genes, each with similar gene structure to the Dag1 genes of bony fish  four additional introns  split the CDS  and in species representative of other arthropod classes (Crustacea and Chelicerata) (Table C. elegans (nematode) includes multiple introns that interrupt the region encoding the IG2 domain and the cytoplasmic domain, respectively  interrupt the N-terminal encoding sequences in a position similar to the additional introns in the DG gene of B. floridae  in which six repetitions of IG2_MAT_NU are present [In our prior study of the molecular phylogeny of DG protein, we identified that the region including the IG2_MAT_NU domains has been independently duplicated in a number of phyla . This phenomenon is particularly striking in the DG of the sea anemone  present . The curA. queenslandica (Porifera) and 48 bp in Capitella teleta (Annelida) and yet comprises up to ~30 kb in Ornithorhynchus anatinus and other mammals , in C. teleta (annelid) and in T. castaneum (insect)  introns. These two introns are located within the 5′ portion of the gene, making the 3′ region essentially intron-less , arthropods (in particular Drosophila melanogaster) and nematodes  includes multiple introns. Larger genomes generally contain genes with longer introns [In vertebrates, a striking characteristic of  introns  and inde introns . A tende introns , 34. The introns . Interes introns . However introns .We found that the observed repetitions of the intronless IG2_MAT_NU module in some species  do not iThe biological significance of the IG1 domain for DG function has been underscored by the recent identification of two novel compound heterozygous DG missense mutations, V74I and D111N, that are associated in a patient with asymptomatic hyperCKemia and hypoglycosylation of α-dystroglycan . The mutD. melanogaster DG (miR9a (CCAAAGA) in myotendinous junction and miR310 s (UGCAAUA) in the brain) [Homo sapiens , Mus musculus  and Hydra magnipapillata . This conservation might indicate that some of the regulatory mechanisms observed in Drosophila melanogaster might also be relevant to other species.Further work will be needed to analyse the 5′ and 3′ untranslated regions (UTR) of dystroglycan genes for possible conserved transcription factor binding sites and/or other regulatory elements such as miRNA hybridization sites , 40. A pe brain) , 42 are Pleurobrachia bachei and Mnemiopsis leidyi), and phylum Ctenophora is not included in the model. Our previous study of the molecular phylogeny of the DG protein demonstrated that the IG2_MAT_NU region and the domains of β-dystroglycan are the most highly conserved regions that might reflect the ancestral form of DG [A. queenslandica DG indicates that the IG1 domain and IG1-intron have been part of the DG gene from its earliest origin. In contrast, the S6 domain appears to have been gained in the last common ancestor of placozoans, cnidarians and bilaterians, perhaps by exon shuffling . Searches were completed as of the end of January 2016. Searches with protein sequences were performed by BLASTP at NCBI Genbank at default parameters and were based on the protein sequences studied in [All the gene sequences investigated were retrieved either from the Ensembl database  or from udied in . Dystrogudied in .http://www.ebi.ac.uk/Tools/msa) and are presented in BoxShade 3.21 (http://www.ch.embnet.org/software/BOX_form.html). Secondary structure elements are reproduced from PDB 1U2C [Multiple sequence alignments of nucleotide or protein sequences were constructed in MUSCLE 3.8  via the PDB 1U2C .Graph presentations and fitted lines were generated using KaleidaGraph (Synergy Software).


The detection of faked identities is a major problem in security. Current memory-detection techniques cannot be used as they require prior knowledge of the respondent’s true identity. Here, we report a novel technique for detecting faked identities based on the use of unexpected questions that may be used to check the respondent identity without any prior autobiographical information. While truth-tellers respond automatically to unexpected questions, liars have to “build” and verify their responses. This lack of automaticity is reflected in the mouse movements used to record the responses as well as in the number of errors. Responses to unexpected questions are compared to responses to expected and control questions . Parameters that encode mouse movement were analyzed using machine learning classifiers and the results indicate that the mouse trajectories and errors on unexpected questions efficiently distinguish liars from truth-tellers. Furthermore, we showed that liars may be identified also when they are responding truthfully. Unexpected questions combined with the analysis of mouse movement may efficiently spot participants with faked identities without the need for any prior information on the examinee. The use of faked identities is a very common issue. People can fake their personal information for a number of reasons. Faked autobiographical information is, for example, observed in sports, with players claiming to be younger than what they really are . Social From the beginning, starting with the pioneer work of Benussi , the ideThe Odyssey, told Polypheme that his real name was “No-man.” This lie was intended to fool Polypheme but was also supposed to be easily spotted as a lie by Polypheme’s one-eyed companions.By contrast, it could be very high when fabricating complex lies, such as when Ulysses, the hero of RT-based memory detection has a number of advantages over alternative psychophysiological techniques, especially when a high number of subjects are under scrutiny. First, RTs are less sensitive to strong individual or environmental changes, such as in the case of physiological parameters. Secondly, this technique has the unparalleled feature that it may be applied using merely a computer and administered to a large number of examinees over the Web. Currently, two memory-detection techniques based on RTs that are used to present words or sentences may be adapted as tools for identity verification. The Concealed Information Test (CIT-RT)  and the The CIT-RT is a technique that consists of presenting the critical information within a series of very similar, noncritical sources of distractor information. For example, if the concealed knowledge about a murder weapon is under scrutiny, a knife (the known murder weapon) will be presented together with distractors that are also potential murder weapons . For the innocent subjects, the response is expected to be similar to all stimuli. By contrast, for the guilty subject (with guilty knowledge for the weapon), longer responses for the critical item are expected . When applied to verify whether the autobiographical information that the examinee claims corresponds to the true identity, the CIT efficiently succeeds in distinguishing the identities of liars and truth-tellers .I am in front of a computer”) or certainly false  for the respondent and related to the moment of testing. The other two categories are represented by alternative versions of the autobiographical memory under investigation , with only one of the two being true. During the test, the examined subject performs a categorization task. The true autobiographical event is identified because it determines faster RTs when sharing the same motor response with certainly true sentences [The aIAT is a memory-detection methodology that exploits consistency/inconsistency between sentences. It includes stimuli belonging to four categories: two of them are logical categories represented by sentences that are certainly true . Available techniques cannot be used when the critical information that is evaluated for veracity  is not available.Here, we will present a new paradigm that overcomes the caveats of the available methods and may be used to identify whether personal information is true. Most importantly, we will show that a faked identity can be spotted in the absence of any information about the suspect’s true identity. Faked identities will be detected using unexpected questions combined with an analysis of mouse movements during the response in a binary classification task. We will show that the analysis of mouse dynamics efficiently detects whether the personal information that the examinee claims is true. In the experiments presented here, the participants do not respond by pressing YES/NO buttons using the keyboard, as in the RT-CIT or aIAT, but they are instead required to respond by clicking with a mouse virtual buttons appearing on the computer screen along with questions regarding their identities. The use of a mouse for recording responses has a number of advantages over the use of a keyboard. While the press of a button may permit only RTs to be recorded, mouse recording allows several indicators to be collected, including but not limited to RT . The technique is also promising regarding resistance to countermeasures, as a high number of movement parameters seems, in principle, more difficult to control entirely via efficient, planned countermeasures to lie detection.It has been shown that the analysis of mouse trajectories can capture cognitive complexity in stimulus processing when participants are required to deliver multiple-choice responses. This procedure has been applied to a large number of fields and has proved useful in highlighting cognitive complexity related to negative sentence verification , racial Here, we will present the results of an experiment in which the trajectories of motor responses using the mouse were investigated while the participants were tested on questions regarding their identities. Two types of questions were asked: expected questions and unexpected questions . Vrij anThe experiment reported here consists of a binary classification task involving expected and unexpected questions about identity. Expected questions covered typical information as reported in documents, while unexpected questions covered information that was well known and automatically retrieved by the truth-teller but that should be “computed-on-the-spot” by liars. An example of an expected question would be one’s date of birth, and a corresponding unexpected question would be the zodiac corresponding to the date of birth. While truth-tellers easily verify questions involving the zodiac, liars do not have the zodiac immediately available, and they have to compute it for a correct verification. The uncertainty in responding to unexpected questions may lead to errors. Furthermore, we found that the trajectory mouse response, analyzed using kinematic parameters and other spatial and temporal parameters intended to capture the uncertainty in motor response, could be useful in detecting deception. Deception, therefore, is expected to reflect itself in the form of the trajectories.In an identity verification task, the liars are typically required to learn the autobiographical information of a new identity and to take the test responding as if that information were real for them. For example, Verschuere et al.  asked su“Unexpected questions will increase a liar’s cognitive load” [For a truthful responder, unexpected questions are supposed to elicit the correct response automatically. By contrast, an identity liar has to reconstruct the non-rehearsed unexpected information and verify it. Therefore, this process takes time before the response is emitted, which is reflected in longer RTs. In short, ve load”  and thisIn the following, we will describe in detail the experiment structure and the measures collected. The ethics committee for psychological research of the University of Padova approved the experimental procedure.Forty Italian-speaking participants were recruited at the Department of Psychology of Padova University. The sample consisted of 17 males and 23 females. Their average age was 25 years (SD = 4.6), and their average education level was 17 years (SD = 1.8). All of the participants were right handed. These first 40 participants were used to develop the model that was later tested, for generalization, in a fresh new group of 20 Italian-speaking participants (10 liars and 10 truth-tellers). This second sample consisted of 9 males and 11 females. Their average age was 23 years (SD = 1.5), and their average education level was 17 years (SD = 0.83). Both groups of subjects provided informed consent before the experiment.Thirty-two sentences displayed in the upper part of the computer screen were presented to all of the participants. The squares representing the YES and NO responses were located in the upper left and upper right of the computer screen. Sixteen sentences required a YES response, and 16 required a NO response, for both the liars and the truth-tellers. The 32 experimental questions were preceded by 6 training questions (3 requiring a YES response and 3 requiring a NO response) on issues related to the identity not included in the experiment proper . Sentences that required a YES response belonged to the following categories:Expected questions: These included information that was rehearsed before the experiment, both for the truth-tellers and for the liars. The liars responded with personal information about fake identity profiles that the experimenter had assigned to them. The truth-tellers responded to questions regarding their true identities.Unexpected questions: The unexpected questions included information closely related to the false identities but not explicitly rehearsed before the experiment by either the truth-tellers or the liars. In this case, the liars responded to information related to the fake identities assigned to them, while the truth-tellers responded to the questions regarding their true identities.n = 8; 4 requiring a YES response and 4 a NO response) included personal information to which the subjects had to respond truthfully because they could not be hidden to the examiner supervising the test. For example, “Are you male?”  required a YES response, whereas “Are you a female?”  required a NO response. Therefore, the control questions required truthful responses by both the liars and truth-tellers, even if they were related to identity.Control questions: Control questions were intermixed with the expected and unexpected questions. The control questions (n = 16) required YES responses. By contrast, 16 questions derived from the expected, unexpected, and control questions required NO responses as displayed in For both the liars and the truth-tellers, half of the expected, unexpected, and control questions : the time between the appearance of the question and the beginning of the mouse movementReaction time (RT): the time between the appearance of the question and the virtual button-pressing performed with the mouseMaximum deviation (MD): the maximum perpendicular distance between the actual trajectory and the ideal trajectory (the line connecting the starting button with the expected response button)Area under the curve (AUC): the geometric area included between the actual trajectory and the ideal trajectoryMaximum deviation time (MD-time): the time taken to reach the point of maximum deviation from the ideal trajectoryx-flip: The total number of changes in directions of the mouse during the full trajectory on x-axisy-flip: The total number of changes in directions of the mouse during the full trajectory on y-axisn, Yn): the position of the mouse along the axis over timeX, Y coordinates over time , Reaction Time (RT), Maximum Deviation (MD), Area Under the Curve (AUC), Maximum Deviation time (MD-time), x-flip, y-flip, Y30, Y29, Y18, Y30–Y29, and Y29–Y18. For each of the variables we computed the average value of the 32 responses for each participant.pb = 0.68), AUC (rpb = 0.53), MD-time (rpb = 0.45), and Y29 (rpb = 0.42) .A correlation analysis was conducted to highlight the independent variables that had the maximum correlation with the dependent variable (truth-tellers vs. liars) and minimum correlation across the independent variables . We consIn this section, the steps followed to analyze the data and the procedure used in developing the ML classifiers are reported.Data and instructions to replicate the results are available as Supporting Information see  Text.The first analysis compared the responses of liars and truth-tellers by averaging across individual responses to both YES and NO responses. In Here we report examples of individual mouse trajectories in response to control questions and unexpected questions collected from a prototypical truth-teller  and a prTrajectories refer to responses to single questions. Note that this liar is responding truthfully to control questions. Nonetheless, his response diverges from the direct trajectory that ideally characterizes a truthful response see . This geWe analyzed the subjects’ performances separately for control, expected, and unexpected questions. In t-tests on the whole sample were carried out in order to compare left and right responses. We did not find any statistically significant difference both for MD-time  and Y29 . For AUC, we obtained the following results: t = -2.09 and p = 0.04, but the Cohen’s d value showed a small effect size (d = -0.33), and the Bayes Factor approached (BF = 1.2). In We investigated whether there is a difference in the trajectory and in response time between the questions to which subjects responded by moving a mouse to the right (questions requiring a NO response) and questions to which subjects responded moving a mouse to the left (questions requiring a YES response). The t-test, Cohen’s d and Bayes Factor.Feature selection isolated, from an original set of 13 predictors, 4 independent variables: errors, AUC, MD-time, and Y29. These were highly correlated with the group (truth-teller/liar). The following table see  reports Several machine learning (ML) classifiers were tested using a 10-fold cross-validation procedure as implemented by WEKA . We seleAs reported in After the development of the ML classifiers described above, a further sample of 20 participants (10 liars and 10 truth-tellers) was collected and tested using the models previously developed based on the original 40 participants. This group of participants was a totally new group that had never been used before for the analysis or model building. This procedure is regarded as an optimal strategy to avoid overfitting . The clTo better understand the contribution of control, expected, and unexpected questions in the classification we ran three separate models for each type of question. Results indicate that the major contribution derives from unexpected questions see . ClassifWe also investigated the relative weight of the predictors by eliminating the independent variables one by one and rerunning the classifiers. The results indicated that after eliminating the errors from the predictors, the classification accuracy dropped to around 75% for the cross-validation and around 70% for the test procedure . The major contribution in prediction accuracy comes from revealing errors to unexpected questions with mouse dynamic features fine tuning an already good classification. This is clear if we consider that predictions based solely on errors yielded the following results: Random Forest: cross-validation = 77.5%, test = 100%; Logistic: cross-validation = 82.5%, test = 100%; SVM: cross-validation = 80%, test = 95%; LMT: cross-validation = 85%; Test = 100%. After dropping AUC from the predictors, the classification accuracy remained stable in the test set and fell to 90% during cross-validation . Similar results were obtained when removing MD-time from the predictors . Finally, after discharging Y29 from the predictors, the accuracy both in the training and the test sets decreased slightly .th time frame.Briefly, the relative importance of the independent variables indicated that the total number of errors gave the major contribution in correctly distinguishing liars from truth-tellers, followed by the MD-time, the AUC, and the position of the mouse along the y-axis on the 29The errors to control and expected questions are virtually absent in truth-tellers see . Liars at = - 4.59, p<0.01; Cohen’s d = 1.60; BF = 16.42.Liars and truth-tellers make no errors to control questions and only a total 2/240 to expected questions. The difference between the two groups arises from unexpected questions with truth-tellers making a total 5/240 errors and liars 82/240. This indicates that for every error made by a truth-teller to unexpected questions liars make 16 errors. It is worth noting that liars make more errors to unexpected YES (60/120 where they lie) rather than unexpected NO (22/120 where they respond truthfully), n = 40 and German n = 20) with results for liars of t = -1.4, p = 0.17  and results for truth-tellers of t = 0.66, p = 0.52 .To check whether the model can efficiently classify participants from different cultures, we tested 20 German subjects (10 liars and 10 truth-tellers) with good results. To address the effects of culture on the generalization of results, we tested a sample of 20 participants native speakers of German in Düsseldorf  with questions in German. Participants provided informed consent before the experiment. Results from this group were evaluated using the model originally trained on the 40 Italian participants. The classification accuracy was the following: Random Forest = 95%, Logistic = 100%, SVM = 90%, LMT = 95%. Errors analysis see  indicateThe experimental design described in the manuscript requires that liars lie only when responding YES to expected and unexpected YES questions. In all of the other conditions , the liars responded truthfully see . An inteIn order to evaluate whether the trajectories of the liars also differed from those of the truth-tellers when they were not lying, we compared the two experimental groups on the independent variables previously used in developing the classifiers. The results of the independent t-test, reported in Both statistical analysis and ML analysis have shown that the markers of lying extended to questions to which they responded truthfully. Even when responding truthfully, the liars could be identified, but with lower accuracy. From a cognitive point of view, what is interesting here is that, in the experimental design, the mind-set of the liars also extended its effects to questions when they were responding truthfully. To our knowledge, this pattern of results has never been reported before and could be an indication of the level of sensitivity of the technique of mouse-movement analysis.To our knowledge, no techniques can accurately spot whether a subject’s ID is true or false without any information about the respondent’s true identity. In this paper, we report results on a new memory detection technique aimed at classifying whether an ID is true or faked when liars do not provide any personal information that is then included in the test itself.The participants responded using a mouse to questions regarding ID that required a YES/NO response. Mouse dynamics provide a rich source of data, as compared to similar binary classification tasks based on response buttons. While the data collected through button presses are limited to recording latency between the question onset and the button press, mouse response permits several parameters to be collected, including reaction times but also initiation time, velocity, acceleration, and the mouse’s trajectory.In order to develop a model that efficiently spots participants with faked identities, we tested the responders with questions that were expected and that liars over-learned in a preliminary learning phase . Together with expected questions targeting the ID document information, a set of unexpected questions related to the expected questions was also presented. Consider, for example, the place of birth. Expected questions that would appear on the ID card would be “Were you born in Pisa?” (requiring a YES response) or “Were you born in New York?” (requiring a NO response). Corresponding unexpected questions would be: “Is Florence the capital of your region of birth?”  and “Is Venice the capital of your region of birth?” . Another unexpected question related to the date of birth (derived from the date) was about zodiac. Truth-tellers are supposed to be able to retrieve the responses about their true zodiac more automatically than liars; therefore, their response is expected to be more rapid, with less errors, and characterized by a more direct mouse trajectory. In general, unexpected questions are supposed to be rapidly retrieved by truth-tellers while liars have to mentally “compute” the response from the original expected information .The research reported here demonstrated that mouse dynamics analyzed using a ML model yielded a correct classification of liars and truth-tellers with more than 90% accuracy. This result was achieved by developing a set of classifiers with comparable performance in the accuracy range 90–95% . Another group was collected and tested (10 truth tellers and 10 liars) to validate the model’s generalization. In this group, the accuracy was confirmed to be comparable to that of the group used for developing the classifiers (95% = 19/20 participants correctly classified), showing that the high accuracy achieved in the model-building stage was not the result of overfitting.Game theory is also a promising technique in deep learning. We did not evaluate whether more complex deep learning models based on game theory concepts –37 couldth time frame.We conducted an analysis to identify the most important predictor, which was total errors followed by the MD-time, the AUC, and the position of the mouse along the y-axis on the 29From a cognitive point of view, it is confirmed that unexpected questions may be used to uncover deception. The power of unexpected questions has been extensively examined in investigative interrogations . Here, wIt is interesting to note that our experimental design requires liars to respond truthfully to a number of questions. The analysis performed on such truthful responses indicates that liars are still detectable, even if with a lower accuracy, when they are not lying. Rosenfeld et al. showed that truth telling liars could be identified using P300, similarly to what we report here . It is iUnexpected questions require answers to be carefully crafted and this may be a limitation in online automatic usage of the technique. Further limitations of the present study include the fact that the procedure has been tested on participants of a single culture and generalization tested on participants belonging to a not so different culture (Germany). A further limit of the present research comes from the fact that the issue of faked ID detection does not permit a direct comparison with more validated lie detection techniques . Any comparison between techniques is therefore only indirect.With all these limitations in mind, we think that the use of unexpected questions combined with the analysis of mouse dynamics seems to be a promising avenue for uncovering deceptive responses.S1 Text(PDF)Click here for additional data file.S2 Text(PDF)Click here for additional data file.S1 Dataset(ZIP)Click here for additional data file.S2 Dataset(ZIP)Click here for additional data file.


The programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway inhibits the activation of T cells and plays a crucial role in the negative regulation of cellular and humoral immune responses. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. In the present study, we aimed to detect the expression of PD-L1 in DLBCL and to analyze its relationship with prognosis.We reviewed medical records of 204 newly diagnosed DLBCL patients in Sun Yat-sen University Cancer Center between October 2005 and August 2012. The expression of PD-L1 in tumor tissues from these 204 patients was detected using immunohistochemical (IHC) assay. The expression of anaplastic lymphoma kinase (ALK), CD5, CD30, and C-Myc in tumor specimens from 109 patients was detected using IHC, and Epstein–Barr virus (EBV)-encoded RNAs (EBERs) were detected using fluorescence in situ hybridization. The Spearman method was used for correlation analysis. The Kaplan–Meier method with log-rank test was used for univariate analysis. Cox proportional hazards model was used for multivariate analysis.P = 0.02 and P = 0.04). Patients with PD-L1 expression in tumor microenvironment were more likely to be resistant to first-line chemotherapy when compared with the patients without PD-L1 expression in tumor microenvironment (P = 0.03). PD-L1 expression in tumor microenvironment was negatively correlated with C-Myc expression . No correlations were detected between PD-L1 expression and the expression of ALK, CD5, and CD30 as well as EBERs. The 5-year overall survival (OS) rates were 50.0% and 67.3% in patients with and without PD-L1 expression in tumor cells (P = 0.02). PD-L1 expression in tumor cells was an independent risk predictor for OS (P < 0.01).Of the 204 patients, 100 (49.0%) were PD-L1-positive in tumor cells and 44 (21.6%) were PD-L1-positive in tumor microenvironment. PD-L1 expression in tumor cells and tumor microenvironment were more common in the non-germinal center B-cell-like (GCB) subtype than in the GCB subtype (PD-L1 expression is more common in the non-GCB subtype than in the GCB subtype. PD-L1 expression in tumor microenvironment has a negative correlation with C-Myc. PD-L1 positivity predicts short survival in DLBCL patients. For patients with PD-L1 expression, more strategy such as anti-PD-L1 antibody treatment should be recommended. Programmed cell death-ligand 1 (PD-L1), a member of the B7 family , is an inhibitory ligand expressed on the surface of macrophages, dendritic cells, fibroblasts, and T cells . BindingAberrant PD-L1 expression was reported in a number of human malignancies , 10. EviAs a group of heterogeneous diseases, diffuse large B-cell lymphoma (DLBCL) can be divided into different clinical and pathological subtypes as well as molecular and immunophenotypic subgroups , 30. AnaIt is suggested that coordinate regulation of PD-L1 among tumor cells and tumor-infiltrating macrophages may exist . ResearcSeveral studies have reported the expression of PD-L1 in lymphoma and described its relationship with prognosis , 40, 41.Clinical data were collected from the database of the Department of Medical Oncology, Sun Yat-sen University Cancer Center .The inclusion criteria were as follows: (1) patients with newly diagnosed DLBCL treated at the Sun Yat-sen University Cancer Center between October 2005 and August 2012; (2) patients were diagnosed using biopsy according to the 2001 or 2008 World Health Organization classification; (3) patients were 18 years of age or older; (4) patients had received first-line chemotherapy regimens, such as R-CHOP  or R-CHOP-like regimen, CHOP  or CHOP-like regimen, and MA regimen (high-dose methotrexate and cytarabine) with curative intent; (5) patients were not infected with human immunodeficiency virus and were not with immunodeficiency disease or second tumor; and (6) complete clinical information was available, including follow-up data. The Ann-Arbor staging system and the international prognostic index (IPI) were used for staging evaluation and risk stratification respectively.All tumor specimens were obtained by biopsy or primary tumor resection before chemotherapy, fixed in 10% buffered formaldehyde, and embedded in paraffin. The 5-μm thick, formalin-fixed, and paraffin-embedded sections were mounted on slides. Slides were deparaffinized in a microwave oven with xylene, then washed using alcohol washes of decreasing concentrations  and distilled water. A heat-induced antigen retrieval method was used with sodium citrate buffer solution (pH 8.0). Rabbit monoclonal antibody for PD-L1 (1:50 dilution) was from Cell Signaling Technology . Mouse monoclonal antibody for other markers assessed in the present study, including ALK (1:50 dilution), CD5 (1:100 dilution), CD30 (1:50 dilution), C-Myc (1:50 dilution), were from Zhongshan Jinqiao Biotechnology .Slides were incubated with primary antibodies at 37 °C for 50 min, incubated with secondary antibody at 37 °C for 30 min, and counterstained with hematoxylin. Next, the slides were dehydrated and covered as per routine laboratory protocols. Two independent pathologists reviewed all specimens separately, and a common consensus was reached in all cases. Cases with any tumor cells expressing CD5 or ALK were considered CD5- or ALK-positive; cases with at least 30% of tumor cells expressing C-Myc were considered C-Myc-positive; cases with at least 20% of tumor cells expressing CD30 were considered CD30-positive. Cases with at least 5% of lymphoma cells showing distinct membranous and/or cytoplasmic staining of PD-L1 were considered PD-L1-positive in tumor cells. Cases with at least 20% of all cells  expressing PD-L1 were considered PD-L1-positive in tumor microenvironment. The results of BCL2 and BCL6 expression were collected from clinical records of the patients.EBERs were detected using the peptide nucleic acid fluorescence in situ hybridization detection kit (Zhongshan Jinqiao) following the manufacturer’s protocol. When at least 10% of cells were stained in the nuclei, the case was defined as EBV-positive.After treatment, patients were followed up every 3 months in the first 3 years and every 6 months thereafter. The final date of follow-up was March 31st, 2016. Routine examinations including physical examination, standard laboratory tests, echocardiography, and a whole-body computed tomography (CT) scan or fluorodeoxyglucose positron emission tomography (FDG-PET) were performed during follow-up. Overall survival (OS) was defined as the duration from pathological diagnosis to death or the last follow-up. Progression-free survival (PFS) was defined as the duration from pathological diagnosis to death, disease progression, or the last follow-up. Patients without any event at the last visit were censored.Chi square test or Fisher’s exact test was used to analyze the relationship between PD-L1 expression in tumor cells/tumor microenvironment and clinicopathological characteristics of DLBCL patients. Pearson’s test was used to analyze the correlation between indexes. The Kaplan–Meier method was used to establish survival curves, and log-rank test was used for univariate analyses. The Cox proportional hazards model was used for multivariate analysis. All statistical analyses used 0.05 as the significance level (two-sided test) and were performed by using SPSS version 19.0 .Clinical data of 204 patients with DLBCL were included in our analysis. The detailed baseline characteristics according to PD-L1 expression in tumor cells and tumor microenvironment are shown in Table The expression statuses of ALK, CD5, CD30, C-Myc, and EBERs were only detected in samples from 109 patients because of the insufficient amount of pathological specimens. ALK was only detected in 1 (0.9%) patient; CD5 was positive in 9 (8.3%) patients; CD30 was positive in 16 (14.7%) patients; C-Myc was positive in 18 (16.5%) patients; C-Myc and BCL2 co-expression was observed in 17 (15.6%) patients; and C-Myc and BCL6 co-expression was observed in 9 (8.3%) patients; 9 (8.3%) patients were EBER-negative.P = 0.02). PD-L1 expression in tumor microenvironment was associated with the non-GCB subtype (P = 0.04), elevated β2-MG level (P = 0.01), and resistance to first-line chemotherapy (P = 0.03). The expression level of PD-L1 in tumor microenvironment had a negative correlation with that of C-Myc . No correlation of the expression level of PD-L1 in tumor cells with that of C-Myc was observed. There was no significant association of PD-L1 expression in either tumor cells or microenvironment with CD5, CD30, C-Myc, and EBERs.PD-L1 expression in tumor cells was associated with the non-GCB subtype (P = 0.02) and 5-year PFS rate  than did patients without PD-L1 expression in tumor cells; the 5-year OS and PFS rates were similar between patients with and without PD-L1 expression in tumor microenvironment  level higher than 245 U/L, failure to achieve complete remission (CR) after first-line chemotherapy, and PD-L1 expression in tumor cells were significantly associated with short OS and PFS; β2-MG level higher than 2.52 mg/L and lack of rituximab treatment were only significantly associated with short OS , and PD-L1 expression in tumor cells  were independent risk factors for OS; stage  was an independent predictor for PFS  1.38; 95% confidence interval (CI) 1.69–9.40; In the present study, we investigated the expression of PD-L1 in tumor cells and in tumor microenvironment in DLBCL patients. PD-L1 expression in tumor cells was significantly associated with poor prognosis. We also found that PD-L1 expression in tumor microenvironment was associated with resistance to first-line chemotherapy and the expression level of PD-L1 in tumor microenvironment was negatively correlated with that of C-Myc. Furthermore, the non-GCB subtype was associated with PD-L1 expression in either tumor cells or tumor microenvironment.PD-L1 expression in B-cell lymphomas is uncommon . GeneralWe set the threshold of at least 5% of lymphoma cells with PD-L1 expression for PD-L1 positivity in tumor cells and at least 20% of malignant and non-malignant cells with PD-L1 expression for PD-L1 positivity in tumor microenvironment, conforming to the cut-point used in a previous publication . We obseP < 0.01), but not for PFS , in multivariate analysis. These results were in accordance with the findings by Kiyasu et al. [P = 0.0060). In the present study, PD-L1 expression in tumor cells and in microenvironment were associated with the non-GCB subtype (P = 0.02 and 0.04), which typically indicated a poor overall prognosis. The mechanisms responsible for the relationship between PD-L1 expression and poor prognosis are still not clear. In classical Hodgkin’s lymphoma, 9p24.1 amplification [In the present study on 204 DLBCL patients, PD-L1 expression in tumor cells was an independent risk factor for OS . C-Myc is a known oncogene in DLBCL. C-Myc gene translocation is a hallmark of Burkitt lymphoma and was detected in 5%–17% of DLBCL patients [C-Myc aberrations include gene translocation, gene amplification, and mRNA or protein overexpression. Duranpanteix et al. [PD-L1 mRNA expression. The regulatory region of PD-L1 gene does not contain a binding site for C-Myc, whereas STAT1 and interferon regulatory factor 1 (IRF1) directly bind to the promoter of PD-L1 gene to increase its transcription, suggesting that C-Myc would repress PD-L1 expression at the mRNA level via STAT1 inhibition [C-Myc regulated the antitumor immune response through binding the promoter region and activating the transcription of CD47 and PD-L1, and C-Myc appeared to initiate and maintain tumorigenesis in a mouse model of Myc-induced T-cell acute lymphoblastic leukemia. Inactivation of Myc could result in the down-regulation of PD-L1, thus restoring recruitment of T cells and macrophages and causing tumor shrinkage. Besides, Rossille et al. [Myc-driven malignancies or even high-grade B-cell lymphomas, especially in those with Myc rearrangement, such as double-hit or triple-hit lymphomas, Myc- and PD-L1-targeted combination therapy may be of potential therapeutic benefit.In the present study, we found that PD-L1 expression in tumor microenvironment had a negative correlation with that of C-Myc (patients . C-Myc ax et al.  investighibition . The mechibition  have shoe et al.  showed tSeveral limitations should be noted when interpreting the results of the present work. As a retrospective study, the long time span for the storage of paraffin-embedded samples may affect the results of PD-L1 detection. The missing clinical data may also affect the accuracy of statistical analysis. Therefore, a study with a larger population is needed to verify our findings. It remains unclear whether the expression of PD-L1 is a key factor associated with the clinical prognosis of DLBCL patients treated with PD-1/PD-L1 blockade therapy. Further integration of genomic and clinical data is expected to deepen our understanding of PD-L1 in DLBCL.In conclusion, the expression of PD-L1 in both tumor cells and microenvironment is associated with the non-GCB-subtype DLBCL. PD-L1 expression in tumor microenvironment has a negative correlation with the expression of C-Myc, which indicates a role of C-Myc in the regulation of PD-L1 expression. PD-L1 predicts short survival in DLBCL patients. For patients with PD-L1 expression in tumor cells, more strategy such as anti-PD-L1 antibody treatment should be recommended.