TrkB expression was detected in 63 (91.3%) out of 69 surgically resected primary GBC specimens by immunohistochemistry. TrkB expression in the invasive front correlated with T factor (p=0.0391) and clinical staging (p=0.0391). Overall survival was lower in patients with high TrkB expression in the invasive front than in those with low TrkB expression (p=0.0363). In vitro experiment, we used five TrkB-expressing GBC cell lines with or without K-ras mutation. TrkB-mediated signaling increased proliferation and the invasiveness by inducing epithelial mesenchymal transition, and activating matrix metalloproteinases-2 (MMP-2) and MMP-9. Inhibition of TrkB-mediated signaling also decreased hypoxia-inducible factor-1α, vascular endothelial growth factor A (VEGF-A), VEGF-C, and VEGF-D expression. In vivo experiment, inhibition of TrkB-mediated signaling suppressed tumorigenicity and tumor growth in GBC. These findings demonstrate that TrkB-mediated signaling contributes to the induction of malignant phenotypes  in GBC, and could be a promising therapeutic target regardless of K-ras mutation status.This study aims to demonstrate the clinical and biological significance of Brain derived neurotrophic factor (BDNF)/Tropomyosin-related kinase B (TrkB) signaling in gallbladder cancer (GBC) through a series of  Bile tract cancer (BTC) is the sixth leading cause of cancer death in Japan, and has been reported more frequently in Japan compared with other countries . The BTCTropomyosin-related kinase B (TrkB), a member of the neurotrophin receptor family, acts as a receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-4/5, and neurotrophin-3 –8, no re2 analysis. Importantly, patients with high TrkB expression in the invasive front had significantly poorer survival than those with low TrkB expression , whereas no staining was observed in normal gallbladder tissue Figure . We foun.3%, wherin vitro. We first examined the role of BDNF/TrkB signaling on GBC proliferation using recombinant human BDNF (rhBDNF) and the Trk tyrosine kinase inhibitor K252a. The phosphorylation of TrkB in GBC cells was observed, as shown in Figure We hypothesized that BDNF/TrkB signaling may contribute to induction of the malignant potential of GBC. We therefore examined BDNF and TrkB expression in 5 GBC cell lines: TGBC2TKB, NOZ, GBd15, TYGBK-1, TYGBK-8. TrkB and BDNF expression were found in all 5 GBC cell lines . On the We next analyzed the effect of BDNF/TrkB signaling on GBC invasiveness. Addition of rhBDNF significantly increased Matrigel invasion by all of the cell lines tested in a dose-dependent manner . K252a tAmong the 5 GBC cell lines that we studied, NOZ is known to exhibit K-ras mutation. In TGBC2TKB and TYGBK-1 cells, addition of rhBDNF induced MEK phosphorylation, and K252a treatment abrogated this effect; whereas in NOZ cells, MEK phosphorylation status was unchanged by BDNF or K252a treatment Figure . TrkB phEMT is considered to be an important factors involved in cancer invasion . TherefoSecond, to determine whether these morphological transformations represent EMT, we analyzed the expression of E-cadherin, vimentin, and several EMT transcription factors  by western blotting. rhBDNF exposure enhanced the expression of vimentin and EMT transcription factors , and these increases in expression were inhibited by K252a. The decrease in E-cadherin expression caused by rhBDNF treatment was abrogated by K252a. Conversely, enhanced expression of vimentin in response to rhBDNF treatment was abrogated by K252a Figure . ConsistThese results demonstrate that BDNF/TrkB signaling induces EMT by upregulating transcription factors in GBC.Matrix metalloproteinases (MMPs) are known to be key enzymes involved in degradation of the extracellular matrix . PreviouA previous report indicated a relationship between BDNF/TrkB signaling and levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) . We therin vitro are reflected in vivo, NOZ (K-ras mutant) and TYGBK-1 (K-ras wild-type) cells transfected with control siRNA or TrkB siRNA were subcutaneously injected into both dorsal regions of athymic nude mice .We used 5 GBC cell lines: TGBC2TKB, NOZ , GBd15 , TYGBK-1All GBC cell lines were seeded onto 96-well plates at 5000 cells/well and incubated with or without recombinant human BDNF  or the Trk inhibitor K252a  for 24, 48, or 72 h. Cell proliferation was assessed by absorbance  at 492 nm (reference wavelength 620 nm) using Cell Count Reagent SF . Forty-eight hours after TrkB small interfering RNA (siRNA) and BDNF siRNA transfection, the cells were reseeded onto 96-well plates and proliferation rate was measured.5) were placed in the upper chamber with or without recombinant human BDNF or the Trk inhibitor K252a, and subsequently incubated for 18 h. Alternatively, cells were treated for 48 h with TrkB siRNA and/or BDNF siRNA, and placed in the upper chamber and incubated for 18 h. The cells that migrated to the lower side of the filter were fixed and stained with Diff-Quik reagent  and then counted under a light microscope .The invasiveness of the GBC cell lines was assessed by Matrigel invasion assay as described previously . Briefly5 cells per well were transfected for 48 hours using Lipofectamine RNAiMAX  according to the manufacture's protocol.ON-TARGETplus™SMARTpool siRNA targeting TrkB (L-003160), BDNF (L-017626), MMP-2 (L-005959), and MMP-9 (L-005970), and negative control siRNA  were purchased from Dharmacon . Following overnight attachment to a 6-well plate, 3 × 10Western blotting was performed as described previously . The proMMP-2 and MMP-9 enzyme activity was assessed by gelatin zymography as described previously . BrieflyTissue samples were obtained from patients with GBC who underwent resection at the Department of Surgery and Oncology, Kyushu University Hospitals, Fukuoka, Japan between 2001 and 2012. Approval for the use of tissues was obtained from patients in accordance with the Ethical Committees for Clinical Study at Kyushu University. Immunohistochemical staining was performed using 4-μm-thick formalin-fixed, paraffin-embedded tissue sections and primary antibodies for TrkB , MIB-1 , VEGF , or HIF-1α . Endogenous peroxidase activity was blocked for 30 minutes using methanol containing 0.3% hydrogen peroxidase. Sections were incubated with primary antibodies overnight at 4°C, followed by incubation for 40 minutes with the secondary antibodies at room temperature. The reaction products were visualized using diaminobenzidine (DAB). The TrkB staining intensity of each slide was separately scored for the tumor invasion front and the tumor center as follows: 1 researcher and 1 pathologist evaluated the whole section on a blind basis and scored the staining intensity as none (0), weak (1), moderate (2), or strong (3) Figure .Slides with xenograft tumors and GBC cells from patients were immunostained with primary antibodies for TrkB  and HIF-1α , followed by Alexa Fluor 488-labeled and Alexa Fluor 594-labeled secondary antibodies , as described previously .6 cells in Matrigel per mouse; n=4 in each treatment group). Tumor size was measured twice a week, and volume was calculated as follows: A × B2× 0.5, where A is the longest diameter and B is the smaller of the two perpendicular diameters of the tumor. The mice were euthanized when the tumor size reached 2 cm in diameter or if the animals became moribund during the observation period.Five-week-old female athymic nude mice (BALB/c nu/nu) were purchased from Charles River Laboratories Japan  and acclimated for 2 weeks. All experimental procedures were approved by the Animal Care and Use Committee of Kyushu University (permit number: A27-324-0). All mice were housed and maintained in a specific pathogen-free animal facility at Kyushu University, and all efforts were made to minimize the number of animals used and their suffering. All experiments were performed in strict accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). NOZ and TYGBK-1 cells transfected with TrkB siRNA or control siRNA were subcutaneously implanted into the flank of nude mice . A χ


The NMe2-GQD@GFET photodetector exhibits high photoresponsivity and detectivity of ca. 1.5 × 104 A W–1 and ca. 5.5 × 1011 Jones, respectively, in the deep-UV region as short as 255 nm without application of a backgate voltage. The feasibility of these flexible UV photodetectors for practical application in flame alarms is also demonstrated.Nitrogen-functionalized graphene quantum dots (NGQDs) are attractive building blocks for optoelectronic devices because of their exceptional tunable optical absorption and fluorescence properties. Here, we developed a high-performance flexible NGQD/graphene field-effect transistor (NGQD@GFET) hybrid ultraviolet (UV) photodetector, using dimethylamine-functionalized GQDs (NMe However, the use of pristine graphene in photodetectors, for which spectral colour selectivity is desired, is hindered by its gapless nature, and by its weak and flat light absorption over a broad wavelength range. To overcome this shortcoming, the fabrication of hybrid photodetectors that can combine the advantages of graphene and a semiconductor material with a tunable bandgap presents the most promising approach. Hybrid graphene photodetectors with various photo-absorbing media have been studied for detection of ultraviolet (UV), visible, and near-infrared (NIR) light. An NIR photodetector with a photoresponsivity as high as ca. 106 A W−1 has been fabricated by hybridizing graphene with PbS nanocrystals2. Integration of plasmonic Au nanorods has also provided NIR light detection with photoresponsivity of 4 × 104 A W−1 at 1,310 nm via the hot carrier injection effect3. Visible light detection with photoresponsivity of 180 A W−1 at 400–800 nm was achieved by the combination of graphene with organometallic halide perovskites4. More recently, a hybrid photodetector that exhibits a full-colour optical bandwidth was produced through immobilization of rhodamine-based organic dye molecules with red, green, or blue light absorption profiles onto graphene, whereby photoresponsivity of 103 A W−1 was obtained5. Such photodetectors’ performance for visible and NIR light detection compares favourably with that of the conventional compound semiconductor-based photodetectors9. In contrast to visible and NIR light detection, no efficient visible-blind UV detection photodetector has yet been developed. For instance, hybrid structures that use wide-bandgap semiconductors  enable UV light detection11. However, the ZnO/graphene hybrid photodetectors were not visible-blind. The TiO2/graphene hybrid photodetectors possessed low photoresponsivity of ca. 0.5 A W−1. Their performance is inferior to those of pristine wide-bandgap semiconductor-based visible-blind UV photodetectors13.Graphene has important potential for use in highly efficient photodetectors because of its combination of broadband optical absorption and high carrier mobility4 A W−1 in the deep-UV to NIR wavelength region that uses a 2D/0D graphene hybrid consisting of graphene and nitrogen-functionalized graphene quantum dots (NGQDs) with tunable bandgaps15. The high photoresponsivity was a consequence of strong light absorption and long-life photogenerated carriers in the NGQDs17. In addition to strong light absorption, NGQDs have bandgap tunability that covers a range of absorption from deep-UV to IR. They can thereby be extended to exploit visible-blind UV photodetection. This report describes the performance of a graphene/NGQD hybrid photodetector for visible-blind flexible UV photodetectors. Dimethylamine-functionalized graphene quantum dots (NMe2-GQDs) with a large bandgap of ca. 3.3 eV were used for the NGQD layer to enable visible-blind UV light detection. The photosensitivity and detectivity of the NMe2-GQD@GFET (graphene field-effect transistor) was investigated. The feasibility of these flexible photodetectors for practical application for flame detection was demonstrated.We have also recently produced a high-performance hybrid photodetector with photoresponsivity of 4 × 102-GQDs with an energy gap of 3.3 eV, which enables visible-blind UV light detection. HRTEM observations indicated that the NMe2-GQDs had diameters of ca. 5 nm , which is characteristic of primary dimethyl amines bonded to graphene , presumably because the localized edge states or surface states in the NMe2-GQDs. The edge states or surface states that contribute to light absorption of NMe2-GQDs in the longer wavelength range have little contribution to the photocurrent because the carriers on these states are highly localised. Moreover, they recombine shortly after generation. In contrast, the shorter wavelength light offers sufficient energy to excite the carriers to the LUMO energy level of the NMe2-GQDs, generating an efficient photocurrent18. A response cut-off wavelength was ca. 430 nm. The UV-to-visible rejection ratio  is the photocurrent)13 of the photodetector is over two orders of magnitude (>5 × 102), as shown in the response spectrum, which indicates that the photodetector exhibits a high signal-to-noise ratio. In fact, a negative value of ΔI was detected, as discussed below.The spectral response was measured. It corresponded well with typical absorbance behaviour for NMe2-GQD@GFET hybrid photodetector by measuring the transfer curve upon illumination. Figure VSD = 0.5 V) for the NMe2-GQD@GFET hybrid photodetector and a pristine GFET. The Dirac point  from the pristine GFET was observed at ca. 23.5 V, implying unintentional hole doping from contamination, lithography processes, and/or surface oxygen-related adsorbates19. After hybridization with the NMe2-GQDs, VD shifted to ca. 4.2 V, which suggests that electrons were transferred from the NMe2-GQDs to graphene. This transferral is consistent with the higher work function of NMe2-GQDs. Upon irradiation  of the NMe2-GQD@GFET hybrid photodetector, VD shifts to a lower voltage, which suggests that photoexcited electrons are transferred to the graphene. From the transfer curves measured in the absence of light, it is estimated that the field-effect mobility for holes is ca. 985 and 1,575 cm2 V−1 s−1, respectively, for the GFET and the NMe2-GQD@GFET. The hole-carrier mobility is increased by the incorporation of NMe2-GQDs, which is contrary to previously observed trends for graphene hybrid phototransistors, which exhibit decreased carrier mobility because of disorder or defects induced by hybridization10. The increase in carrier mobility might be attributable to an increase in the density of states near the Fermi level induced by the widely distributed π orbitals of the NMe2-GQDs.We further characterized the NMe2-GQD@GFET is operated at VG = 0 V, the major carriers in the graphene layer are holes. The holes are compensated by the transferred electrons, which engenders decreased conductance and a negative ΔI  were observed. Rext (Rext = ΔI/(PlightAPD/Alight), where Alight is the light spot area) was calculated with a scaling factor (APD/Alight) that takes into account that only a fraction of the optical power density impinges on the photodetector. In the hole-conduction region (VG < 4.2 V), the holes are compensated by the photoinduced electron carriers, which engenders decreased conductance and a negative Rext. In contrast, the photoinduced electron carriers raise the electron concentration, which engenders increased conductance and a positive Rext in the electron-conduction region (VG > 4.2 V).When the NMe ΔI Fig. . The bac ΔI Fig.  is also 2-GQD@GFET hybrid photodetector by measuring its response time. Figure −2 ] + ΔI2[1 − exp(−t/τ2)]20. Time constants τ1 and τ2 are, respectively, 22 s and 77 s. This slow decay is probably associated with the multiplicity of carrier traps in NMe2-GQDs from quantum confinement and different localized states in the π-π* gap21. The short relaxation time τ1 corresponds to the lifetime of the holes trapped in the NMe2-GQDs, whereas the long relaxation time τ2 represents the time duration for the charge transfer and transportation in NMe2-GQDs layer. The carrier transit time τtransit (τtransit = L2⁄μ∙VDS) is estimated as 1.3 ns (based on a carrier mobility (μ) of 1,575 cm2 V−1 s−1, a channel length (L) of 10 μm, and a bias (VDS) of 0.5 V), thereby the photoconductive gain G (G = τlife/τtransit)22 is between 1.7 × 1010 and 5.9 × 1010, respectively using lifetimes of 22 s and 77 s. The high photoconductive gain is attributed to the cooperative effect of long hole-carrier lifetime in the NMe2-GQDs and the high carrier mobility of graphene. Multiple carrier circulation occurs in the graphene channel, depending on the ratio of the photoexcited holes relaxation time in the NMe2-GQDs layer to the carrier transit time in the graphene layer. Because the photogenerated holes remain trapped in the NMe2-GQDs, the positively charged NMe2-GQDs induce negative carriers in the graphene sheet through capacitive coupling. As long as the NMe2-GQDs remain positively charged, negative charges in the graphene sheet are highly recirculated because of their high carrier mobility, resulting in high photoconductive gain2.We investigated the dynamic performance of the NMeRext, and the specific detectivity D* of the NMe2-GQD@GFET hybrid photodetector at a fixed bias of 0.1 V under light irradiation at wavelengths of 255, 370, and 405 nm. D* (D* = (∆f APD)1/2Rext/in, where APD, ∆f and in respectively stand for the area of the photodetector channel, electrical bandwidth, and noise current)24 represents the capability of detecting low-level light signals. The noise current of photodetectors is dominated by the shot noise, but the noise current in the shot-noise limit is given as in = (2eIdark∆f)1/2, where e and Idark respectively denote the electron charge and the dark current24. Therefore, D* in the shot-noise limit is calculable by the expression: RexAPD1/2/(2eIdark)1/2 25, which has been used to estimate the D* of photodetectors in earlier studies27. The NMe2-GQD@GFET hybrid photodetector exhibited high photoresponsivity and detectivity in the deep UV region. Rext and D* respectively reached ca. 1.5 × 104 A W−1 and ca. 5.5 × 1011 Jones at 255 nm. The maximum values of Rext and D* are comparable to those reported for other high performance UV photodetectors  were used as a precursor for preparation of the NGQDs. N,N-dimethylformamide (DMF) as a solvent, and sulfuric acid and nitric acid as oxidizing agents were obtained from Wako Pure Chemical Industries Ltd.2-GQDs were selected for the NGQD layer because their large energy gap (3.3 eV) enables visible-blind UV light detection. The NMe2-GQDs were prepared as follows. Oxidized GNPs (oGNPs) were obtained by refluxing GNPs in a 2:1 solution of concentrated H2SO4 and HNO3. 5 mg of the oGNPs were dispersed in 5 mL of DMF. The mixture was refluxed at 154.5 °C for 5 h. After cooling to room temperature, the suspension was dried using an evaporation system . The dried powder (NMe2-GQDs) was re-dispersed in water and was dialyzed for 3 days. After drying, the NMe2-GQD powder was re-dispersed in DMF.NMeL of 80 μm and a channel width W of 200 μm. NMe2-GQD dispersions were subsequently dropped directly onto the top of the graphene layer.Monolayer graphene sheets formed by chemical vapour deposition (CVD) onto a PEN substrate (188 μm) were obtained from Graphene Platform Corp. Gold source and drain electrodes (100 nm) were fabricated directly on the top of the graphene/PEN substrate using sputter deposition through a metal shadow mask. These devices had effective channel length 2 layer) were obtained from Graphene Platform Corp. The source and drain electrodes were patterned directly on the top of the graphene/Si wafer using a conventional photolithography process with a poly(methyl methacrylate) resist. The contacts were formed with titanium and gold (15 nm/100 nm) using a sputtering deposition method, followed by a lift-off process. These devices were fabricated with L = 10 μm and W = 10 μm. After the gate contact was deposited onto the back side of the Si wafer, the device was diced into 1 × 1 cm substrates. The NMe2-GQD dispersions were dropped directly onto the top of the graphene layer.Measurements of the gate-voltage dependence on the photoresponsivity were taken using photodetectors fabricated by replacement of the PEN substrate with a Si substrate. The gate contact was deposited onto the back side of the Si wafer substrate. Monolayer graphene sheets formed by CVD onto a p-doped Si wafer  with different wavelengths were used as light sources to provide light with wavelengths of 255, 370, and 405 nm. A fibre probe, coupled to a monochromator with a deuterium lamp, was used for spectral response measurements. The optical power density through the aperture (Popt) was measured using a silicon photodetector . The incident (Pinc) power density was then calculated using the relation: Pinc = (Adet × Popt)/A, where Adet is the active area of the photodetector. Electrical measurements were taken using a system source meter  in conjunction with LabVIEW software. All measurements were conducted at room temperature in the ambient atmosphere.A light source was guided into the photodetector through a circular aperture with area α radiation source (100 μm spot diameter) of 1486.6 eV under high-vacuum conditions. Scans were acquired in the fixed analyzer transmission mode with pass energy of 26 eV and a surface/detector takeoff angle of 45°. High-resolution transmission electron microscopy  was used with accelerating voltage of 200 kV. Optical transmittance and reflectance in the visible/NIR region of the samples was recorded using a spectrophotometer . Reflectance was measured at an incident angle of 5° using an additional attachment. Measurements of work function and shallow energy levels were taken using a photoelectron spectrometer . Atomic force microscopy  was used to investigate the height profile.X-ray photoelectron spectroscopy  measurements were performed using a monochromated Al KThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request and with permission of Toyota Central R&D Laboratories, Inc.Supplementary InformationSupplementary Movie


Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. For the development of more effective immunotherapies, it is first necessary to elucidate the immunological escape mechanisms. In this study, we applied our recently developed highly sensitive immunostaining method employing fluorescent phosphor-integrated dot (PID) nanoparticles to evaluate the prevalence of programmed death ligand 1 (PD-L1) in patients with PDAC.This study included 42 patients with PDAC who underwent pancreatectomy. We evaluated PD-L1 expression in these patients using PID staining and correlated PD-L1 expression level with each patient’s clinico-pathological features.P = 0.049]. Among CD8+-tumor-infiltrating lymphocyte-positive cases, the overall survival of PD-L1-positive patients was significantly poorer than that of PD-L1-negative patients . Univariate and multivariate analyses indicated that PD-L1 expression was an independent predictive poor prognostic factor in patients with PDAC.PD-L1 expression was detected in 61.9% (26/42) of the patients with PDAC by PID staining. There was a significant difference in overall survival between PD-L1-positive and PD-L1-negative patients [hazard ratio (HR) 2.07, 95% confidence interval (CI) 1.00–4.54; PD-L1 expression appears to be an important prognostic factor in patients with PDAC who underwent surgical resection. Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a poor prognosis; it is the fourth leading cause of cancer-related deaths in Japan and the fourth most common malignancy in the USA , 2. At tExpression of programmed death-1 (PD-1) is significantly upregulated on activated cancer-specific T cells. The PD-1 receptor attaches to its ligand PD-L1, which is expressed by tumor cells and infiltrating immune cells. The interaction of PD-1 and PD-L1 inhibits T-cell activation and promotes tumor immune escape –7. The eAlthough the efficacy of PD-1/PD-L1 antibody therapy should be correlated with PD-L1 protein expression in tumor cells, approximately 10–40% of PD-L1 immuno-negative cases also respond to anti-PD-1/PD-L1 therapy –11. ThisThe aim of the study reported here was to evaluate the expression of PD-L1 in patients with PDAC by immunostaining using PID technology and to compare the results with those obtained after conventional DAB staining.This study included 42 patients with PDAC, of whom 31 underwent pancreaticoduodenectomy and 11 underwent distal pancreatectomy at the Department of Surgery at Kansai Medical University Hospital  between May 2001 and December 2007. All patients had histologically confirmed PDAC. The tumors were classified according to the TNM classification . The cliSurgically resected specimens were fixed in formalin and embedded in paraffin; and serial sections cut from the embedded specimens were stained with hematoxylin and eosin for histological evaluation. The most representative tumor areas were sampled for the tissue microarray using 2-mm-diameter samples .This study was conducted in accordance with the Declaration of Helsinki, and the study protocol was approved by the institutional review board of our hospital (Protocol no. H151043 and 27-14).Four-micrometer-thick sections were deparaffinized using routine procedures. Endogenous peroxidase activity in the deparaffinized sections was blocked by treating the sections with 3% hydrogen peroxide treatment for 15 min, followed by washing in deionized water for 2–3 min. The sections were subsequently boiled in 10 mM sodium citrate buffer for 10 min at 121 °C, then allowed to cool at room temperature for 40 min, followed by rinsing with deionized water and washing with phosphate-buffered saline for 5 min.The sections were incubated with the primary antibody toward PD-L1 . Sections were incubated with 2 μg/mL biotinylated anti-rabbit antibody (LO-RG-1) for 30 min and then with PID-conjugated streptavidin (0.06 nM) for 2 h, both at room temperature. The sections were then irradiated at 580 nm, and the fluorescence intensities were measured using a BX53 fluorescence microscope ; images were acquired with a DP73 CCD camera (Olympus)  diluted in antibody diluent . The sections were treated with peroxidase-labeled secondary antibody  after linker reagent treatment for 15 min. The sections were then rinsed in the buffer and immersed in DAB to observe color development.For the DAB-naked eye evaluation, three observers  assessed the immunostaining results in a blinded manner without knowledge of the clinical or histopathological diagnoses. Intensity was graded on a 3-tier scale . The percentage of staining was recorded, and a semi-quantitative (H-score) approach  was used+ lymphocytes and PD-L1 staining with the PID method, was performed to count the tumor infiltrating lymphocytes (TILs)   with a 95% confidence interval (CI). P < 0.05 was considered to be statistically significant.Statistical analysis was performed using JMP® 10 software . Student’s The patient background and clinico-pathological parameters are shown in Table Of the 42 patients, 27 (64.3%) underwent pathological curative resection, eight (19.0%) received neo-adjuvant chemo-radiation therapy, and 23 (54.8%) received adjuvant chemotherapy.To judge the rate of positive PID staining, we first established the threshold of the PID staining value. The average of the highest value of the negative control of PID staining was 3.01; therefore, the threshold value for judging positive PID staining was set to 3.0. Using this threshold, we detected PD-L1 expression in 26 of the 42 patients (61.9%).By contrast, PD-L1 expression measured by the DAB-naked eye evaluation was detected in only six of the 42 patients (14.3%). This expression rate was measured to be between the PID positive staining value derived from the threshold set at 4.0  and 5.0 .The correlations between pathological features and PD-L1 expression are shown in Table P = 0.021). There was no significant correlation between PD-L1 expression and tumor size, lymph node metastasis [including distant lymph node metastasis (M1)], pre-operative tumor marker CA19-9 level, and R0/R1  status. CD8+ TIL counts were not significantly correlated to PD-L1 expression.The ratio of males was significantly higher in the PD-L1-positive group  than in the PD-L1-negative group . The MST was 23.5 months in the PD-L1-positive group and 51.6 months in the PDL-1-negative group. Among the 29 patients who had positive CD8+ TILs (>3 cells per 400× field), those in the PD-L1-positive group showed a significantly poorer overall survival rate than those in the PD-L1-negative group based on detection with the PID method .Survival curves obtained using the Kaplan–Meier method are shown in Fig. P = 0.045); specifically, in the 29 TIL-positive patients, PD-L1 expression determined by the PID method was an independent predictive poor prognostic factor (Table P = 0.003).The results of the univariate and multivariate analyses in the 42 patients are shown in Table We report here the results of our investigation on PD-L1 expression in patients with PDAC. The PD-L1-positive detection rate in PID staining was higher than that in DAB staining, possibly due to the fact that digital immunostaining can detect proteins at lower concentrations. The higher fluorescence intensity of the nanoparticles contributes to the higher sensitivity of PID compared to DAB. In addition, quantitative analysis was possible even in cells with strong positive expression, without saturation.In this study, the PD-L1 positive expression rate detected using the PID method in PDAC cases was higher than that reported in previous studies (4–49%) –17 usingImmunotherapeutic approaches, most notably immune checkpoint inhibitors epitomized by antibodies directed against T-lymphocyte regulators, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and PD-1, have demonstrated efficacy in a variety of solid tumors, including metastatic melanoma and lung cancer, and have already received U.S. Food and Drug Administration approval. PD-1/PD-Ll pathway blockade has resulted in significant and durable clinical responses in patients with several malignancies, such as melanoma, renal cell carcinoma, lung cancer, mismatch repair-deficient colorectal cancer, and bladder cancer . However+ TILs. This result suggests that an immunosuppressive tumor microenvironment with high PD-L1 expression can interfere with the attack by TILs on the tumor cells. In these cases, there is a possibility that blockage of the PD-1/PD-L1 signaling pathway can result in the attack of TILs being dramatically more effective. Although there have been no objective responses observed in patients with PDAC who received anti-PD-L1 antibody therapy [In a clinical setting, patients with positive PD-L1 expression tend to show significantly unfavorable outcomes. Previous studies using DAB methods have also demonstrated that patients with PD-L1-positive PDAC showed unfavorable outcomes. Wang et al. reported a correlation between B7-H1 (PD-L1) expression and pathological grade and TNM stage . Nomi et therapy , our resPhosphor-integrated dot staining provided superior results compared to those obtained by the canonical DAB staining method. We have shown for the first time that PD-L1, detected using PID staining, is a novel prognostic marker for human PDAC. Digital immunostaining is a promising tool for companion diagnostics to evaluate the therapeutic effects of molecular-targeted drugs and immunotherapy.


Cryptococcus neoformans and Cryptococcus gattii, which differ in their ecology, epidemiology, and antifungal susceptibility. Therefore, the search for new molecular markers for genotyping, pathogenicity and drug susceptibility is necessary. Group I introns fulfill the requisites for such task because (i) they are polymorphic sequences; (ii) their self-splicing is inhibited by some drugs; and (iii) their correct splicing under parasitic conditions is indispensable for pathogen survival. Here, we investigated the presence of group I introns in the mitochondrial LSU rRNA gene in 77 Cryptococcus isolates and its possible relation to drug susceptibility. Sequencing revealed two new introns in the LSU rRNA gene. All the introns showed high sequence similarity to other mitochondrial introns from distinct fungi, supporting the hypothesis of an ancient non-allelic invasion. Intron presence was statistically associated with those genotypes reported to be less pathogenic (p < 0.001). Further virulence assays are needed to confirm this finding. In addition, in vitro antifungal tests indicated that the presence of LSU rRNA introns may influence the minimum inhibitory concentration (MIC) of amphotericin B and 5-fluorocytosine. These findings point to group I introns in the mitochondrial genome of Cryptococcus as potential molecular markers for antifungal resistance, as well as therapeutic targets.Cryptococcosis, one of the most important systemic mycosis in the world, is caused by different genotypes of  Cryptococcus neoformans and Cryptococcus gattii  for C. gattii.Various molecular techniques have already been applied to the epidemiological study of cryptococcosis , and other birds, whereas C. gattii is frequently isolated from vegetal material, such as Eucalyptus trees , while C. gattii usually infects immunocompetent patients  than C. neoformans  as a possible candidate for this purpose.Although the methods for molecular differentiation are very specific, their application is time-consuming, normally requires multiple steps and equipment, and in some cases, depends on subjective interpretation, such as the methodologies involving sequencing  in the mitochondrial LSU rRNA of Cryptococcus have already been described in a C. neoformans var. neoformans (VNIII genotype) isolate  and also that in vitro antifungal tests indicate a relationship between intron absence and high MIC (minimum inhibitory concentration) values for 5-fluorocytosine.The present study was designed to investigate the occurrence and variability of mitochondrial C. neoformans and C. gattii isolates from different sources and one isolate of their sister species, C. laurentii, were included in the study , 10 μL PCR buffer CG 5X , 5 μL 30% DMSO (Thermo Scientific), 1 μL deoxynucleoside triphosphates , 30 ng of genomic DNA, 1 U taq Phusion DNA Polymerase (Finnzymes), and 1.25 μL of each primer (20 μM) URA5 (5′-ATGTCCTCCCAAGCCCTCGACTCCG-3′) and SJO1 (5′-TTAAGACCTCTGAACACCGTACTC-3′)  and HhaI  for 3 h at 37°C, and the fragments were separated by 3% agarose gel electrophoresis stained with ethidium bromide at 100 V during 90 min. The isolates were classified following the URA5-RFLP pattern proposed by Meyer et al.  and CryLSUR (5′ GGTATATGCATGCTTGACTGC 3′) herein designed for annealing at 5′ and 3′ positions that flank the two introns previously described in the mitochondrial LSU rRNA gene  stained with ethidium bromide at 90 V during 60 min.PCR of IlustraTM GFXTM PCR DNA and Gel Band Purification kit , following the manufacturer's instructions and sequenced at MACROGEN/South Korea. All the sequences were aligned together with reference sequences  using PRANK online (EMBL-EBI) (http://www.ebi.ac.uk/goldman-srv/webPRANK/)  , using the four representative introns sequenced in this study. Sequences with query cover lower than 25% were not included in the study. Sequences were aligned with PRANK on-line (EMBL-EBI) (http://www.ebi.ac.uk/goldman-srv/webPRANK/)    Zuker, . The con) Zuker,  were con) Zuker, . The prein vitro broth microdilution method was carried out using the Clinical and Laboratory Standards Institute (CLSI), guides M27-A3 and M27-S3 , pH 7.6 (buffered with 1M HCl) and sterilized by filtration. Drug dilutions were dispensed in 96-well microdilution plates, with the final concentrations of the drugs ranging from 0.125 to 64 μg mL−1 for 5-fluorocytosine and 0.03125 to 16 μg mL−1 for amphotericin B and itraconazole. Final inoculum concentrations ranged from 0.5 × 103 to 2.5 × 103 CFU/mL in each well containing different concentrations of the antifungal drugs. The quality-control strains, Candida parapsilosis (ATCC 22019) and Candida krusei (ATCC 6258), were used in each test. Drug-free and yeast-free controls were included. Plates were incubated at 35°C up to 72 h.The MICs (minimum inhibitory concentrations) for all strains were read visually 48 and 72 h after yeast inoculum. MIC for amphotericin B was defined as the lowest concentration that caused 100% inhibition of growth compared to the drug-free control well, and MICs for fluconazole and itraconazole as the lowest drug concentration that caused a prominent decrease in growth (50%) compared with the controls.C. neoformans or C. gattii) data were treated as dichotomous variables. Fisher exact tests were used to test for differences in intron distribution between genotype groups . MIC was treated as ordered variable, since it was divided into four categories from lowest to highest MIC. Thus, to assess the effect of introns on fungi response to antifungals drugs, we applied ordered logistic models using MIC as response variable and intron (absent as reference) and species information (C. gattii as reference) as predictors. In addition, we tested the hypothesis that the species modifies the effect of the introns on MIC by including the interaction term (introns*species). We used the minimum Akaike information criterion (AIC) approach to evaluate the model that better fits the data. The predicted probabilities of being in one of the MIC categories were calculated via post estimation command. The statistical analyses were performed in Stata 11.1  and species , followed by C. gattii (n = 26). Overall, the most common molecular types were VNI (39 isolates) and VGII (20 isolates), followed by VNIV (5 isolates), VNII (4 isolates), VNIII (3 isolates) and two isolates each for VGI, VGIII, and VGIV  and Cne.mL2504 (with 417 bp) (http://www.rna.icmb.utexas.edu/). The primers CryLSUF and CryLSUR, herein designed, anneal to the 5′ and 3′ exons flanking these introns. The amplicon is expected to be 300 bp when the introns are absent, and around 1.9 Kb when both introns are present. It was observed that the PCR products ranged from approximately 300 bp to 2.5 Kb among the eight molecular types . The 300-bp sequenced fragment had 99% similarity with LSU rRNA gene of C. neoformans var. grubii (GenBank accession number AY560612).Aligned sequences of the PCR products for mitochondrial C. neoformans/C. gattii species complex, were herein named Cne.mL2439 and Cne.mL2584, in C. neoformans, and Cga.mL2439 and Cga.mL2584, in C. gattii. Cne.mL2584/Cga.mL2584 were found in VNIII, VNIV, VGI, and VGIII genotypes whereas Cne.mL2439/Cga.mL2439 were found in all C. neoformans, except in VNI and in all C. gattii, excluding VGII. We also observed that some C. gattii genotypes contain a group I intron at position 2504, identifying it as Cga.mL2504. This intron was previously described only in C. neoformans strains (Cne.mL2504) . Therefore, introns at one site in the LSU rRNA gene are not invading other sites in the same gene. The elements at the 2504 position form a basal non-monophyletic clade with no apparent divergence between the Cne.mL2504 and Cga.mL2504 introns. While Cga.mL2504 constitute a monophyletic clade, Cne.mL2504 is paraphyletic (it should include Cga.mL2504 introns to be monophyletic), which could indicate that the intron at the 2504 position has been horizontally transferred between species. Introns at the 2449, 2584, and 2439 positions constitute distinct, monophyletic and well-defined clades. Introns at the 2449 site occur only in C. neoformans and are closely related to other introns from other mitochondrial genes, mostly from other basidiomycetes. The 2584 introns also form a monophyletic clade, closer to LSU rRNA introns from other basidiomycetes; however, Cga.mL2584 is paraphyletic because it shares a common ancestor with Cne.mL2584, which is another possible indication of horizontal transfer of these elements between C. neoformans and C. gattii. Cne.mL2439 and Cga.mL2439 are together a monophyletic group, closely related to COX1 introns from an ascomycete and a basidiomycete, with no informative polymorphism to distinguish Cryptococcus species.According to the phylogenetic tree Figure , each ofp-value of 0.063. The overall analysis considered the presence of at least one intron, regardless intron quantity and site insertion in LSU rRNA for each isolate, and it was observed that intron presence is associated with those genotypes reported to be less virulent (p < 0.001). Future work involving virulence assays will be needed to confirm this association between intron presence and low virulence.We detected an association between intronless isolates and those genotypes shown in some reports to be the most virulent and less susceptible to antifungal agents   for the beta coefficient estimate of the intron term. Therefore, based on these selected models, the results indicate: (1) the presence of introns is associated with a higher amphotericin B MIC (p = 0.010); (2) C. neoformans species is associated with a higher amphotericin B MIC (p = 0.016); (3) C. neoformans is associated with a lower itraconazole MIC (p = 0.036); and (4) the presence of introns is associated with a lower 5-fluorocytosine MIC (p = 0.026).Table p = 0.009 for the interaction term). For example, in C. gattii intron presence is more associated with higher MIC values, whereas in C. neoformans it is associated with lower MIC values .Besides these marginal effects, the species clearly modifies the effect of intron presence on the amphotericin B MIC  and were named Cne.mL2439 and Cne.mL2584 in C. neoformans and Cga.mL2439 and Cga.mL2584 in C. gattii. Their predicted secondary structures show the presence of the main helical domains and consensus peripheral elements typical of group I introns, such as the substrate-binding domain (P1 and P10 helices), the P4/P6 scaffolding domain  and the P3/P9 catalytic domain  that contains the guanosine-5′-triphosphate (GTP) binding site within the P7 helix , suggesting that some group I introns remain mobile in the genome by the homing process. HEGs are DNA-cleaving enzymes that recognize long-specific DNA sequences and performs a double-strand break (DSB), promoting the specific duplication of group I introns . After introduction of the DSB in the intronless allele, homologous recombination uses the intron-containing allele as a template for repair  and Amoebidium . This could indicate a possible horizontal transfer. In fact, autocatalytic introns are very ancient and frequently transferred laterally, so that the main hypothesis for their sporadic distribution considers their presence in genomes even before the endosymbiosis event by prokaryotes, which would eventually become intracellular organelles in eukaryotic cells. Indeed the structural similarity between some autocatalytic introns (group II introns) and spliceosomal introns give us some clues about the origin of interrupted genes in eukaryotic nuclear genomes   outbreak with novel, highly virulent strains/subtypes  that dispersed into neighboring regions including the United States . In fact, since the mechanism of action of this drug focuses on the fungal cell membrane, altering its permeability, and not on nucleic acid metabolism as does 5-fluorocytosine, we expected no association between MIC and intron presence, as seen for the itraconazole drug. This may simply suggest that the presence of these introns in some isolates might be in linkage to other genetic markers responsible for these increased MIC values. However, again this is an area for future research on more isolates of both species with specific antifungal tests, as well as in vitro assays on the action of these drugs on intron splicing efficiency.Interestingly, and divergently from the observed effect of 5-fluorocytosine, high MIC values for amphotericin B were associated with intron presence in C. neoformans evaluated here showed higher MIC values for amphotericin B than did C. gattii, which could indicate a peculiarity of these C. neoformans isolates (most from VNI genotype) from Rio Grande do Norte, Brazil, since usually no significant difference is observed for this drug, as previously reported , which could reflect the already observed difference of susceptibility between VNI and VGII , rather than species, is actually the relevant factor for the increased susceptibility to this drug.Concerning the association between species and MIC values for the three drugs tested here, according to the statistical analysis, the Cryptococcus isolates belonged to VNI and VGII genotypes, which are the most frequent ones in Brazil (Santos et al., LSU rRNA genes, and in other mitochondrial genes, as molecular markers for all genotypes of C. neoformans and C. gattii, given the polymorphic characteristic of these elements. Nevertheless, this was the first study in which mitochondrial autocatalytic introns of the LSU rRNA gene from C. neoformans and C. gattii were investigated in a significant number of isolates from different genotypes. Our main findings point to a large polymorphism in group I introns, including the secondary structure prediction of two new introns, as well as to a variation in their presence and nucleotide lengths among the different genotypes of Cryptococcus not previously described. Despite the fact that these introns cannot differentiate completely all the Cryptococcus genotypes, an association was observed between intron absence and the genotypes known as most virulent, such as VGII, VGI, VNI, and VNIV. Moreover, our antifungal susceptibility assay revealed a suggestive relationship between intron presence and susceptibility to 5-fluorocytosine, indicating that these elements might be therapeutic targets, as previously mentioned for C. albicans (Mercure et al., in vitro studies will address the effect of these and other drugs on group I intron splicing in the pathogenic Cryptococcus species.In this study, most of the Cryptococcus neoformans and C. gattii are important worldwide-distributed pathogens that cause systemic mycoses with prolonged treatment and in some cases, depending on their genotype, present a resistance profile to antifungal drugs. Genotype-specific diagnosis using molecular markers for drug susceptibility and virulence may contribute to a correct treatment, improving patient prognosis. Mitochondrial group I introns in LSU rRNA gene from Cryptococcus are good candidates for such markers, since our results showed that their absence seems to be associated with those genotypes shown to be the most virulent and to have high MIC values for 5-fluorocytosine, corroborating their potential as new and alternative drug targets. In addition, the discovery of two new introns in mitochondrial LSU rRNA gene shows that the polymorphism of these elements in mtDNA may be even higher than initially thought. Since they are present in extremely important genes for fungal cell survival, mainly during host-pathogen interactions, evolutionary studies on their distribution dynamics in the mitochondrial genome of C. neoformans and C. gattii might reveal new markers for virulence and drug susceptibility.Cryptococcus and co-wrote the manuscript, RT designed and supervised the experimental procedures as well as organized the writing manuscript.FG did the literature review and most experimental procedures and wrote the manuscript, TA, JF, and HR assisted in phylogenetic, infernal/RFAM and intron structure analysis and co-wrote the manuscript, LF statistically analyzed the results and co-wrote the manuscript, SB, MO, and GD supplied most of fungal culture as well as infrastructure for culturing and DNA extractions of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Graphene displays properties that make it appealing for neuroregenerative medicine, yet its interaction with peripheral neurons has been scarcely investigated. Here, we culture on graphene two established models for peripheral neurons: PC12 cells and DRG primary neurons. We perform a nano-resolved analysis of polymeric coatings on graphene and combine optical microscopy and viability assays to assess the material cytocompatibility and influence on differentiation. We find that differentiated PC12 cells display a remarkably increased neurite length on graphene (up to 27%) with respect to controls. Notably, DRG primary neurons survive both on bare and coated graphene. They present dense axonal networks on coated graphene, while they form cell islets characterized by dense axonal bundles on uncoated graphene. These findings indicate that graphene holds potential for nerve tissue regeneration and might pave the road to novel concepts of active nerve conduits. A specific feature of peripheral nerves is the ability to spontaneously regenerate after traumatic injuries. In the presence of important gaps where an end-to-end suture is not possible, a surgical approach is used, where nerve conduits  are used as bridges between the nerve stumps and provide physical guidance for the axons , carbon nanotubes (CNT), GO and rGO to star in its play  with the ones on 4 possible control substrates: hydrogen etched SiC (SiC), gold coated glass coverslip (Au), glass coverslip (Glass) and polystyrene plate (well). The latter, being routinely used in cell culture procedures, was used as classic control. SiC controls were implemented since graphene was grown directly on such substrates, which display a good biocompatibility    solution in water (Sigma-Aldrich), 200 μg/ml Collagene Type I (Sigma-Aldrich) in DI water, 30 μg/ml Poly-D-lysine (PDL) (Sigma-Aldrich) in PBS, 30 μg/ml PDL and 5 μg/ml laminin (Life Technologies) in PBS. The samples were incubated with the coating solution at 37°C for 1, 4, and 12 h and rinsed three times in DI water before analyzing their topography via AFM. AFM was performed in tapping mode on samples with and without the polymeric coating, over several areas up to 10 × 10 μm wide. AFM micrographs were analyzed using the software Gwyddion 2.45.Figure 2. Cell viability was assessed with the Cell counting Kit-8 assay , based on quantification of WST reduction due to the metabolic activity of viable cells. Samples were prepared according to the manufacturer's instructions and measured at the GloMax® Discover multiplate reader (Promega). The results are reported as % over the polystyrene well, considered as control. All the experiments were repeated at least twice independently.PC12 cells (ATCC® CRL-1721™) were maintained in a humidified atmosphere at 37°C, 5% CO2 in RPMI 1640 medium supplemented with 10% horse serum, 5% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine (Gibco). Cells were plated at ~40–60% confluency onto the substrates previously coated with 100 μg/ml Poly-L-lysine solution (PLL) in water (Sigma-Aldrich). Differentiation was achieved using two different procedures: (1) direct addition of 50 ng/ml NGF (Alomone Labs) in complete cell medium after seeding; (2) a 5–6 days priming with 15 ng/ml NGF in complete medium, followed by seeding on the substrates with 50 ng/ml NGF in RPMI medium supplemented with 1% horse serum, 0.5% fetal bovine serum, 1% penicillin/streptomycin and 1% L-glutamine. In both cases, 2/3 of the medium was renewed every 2–3 days. With the second procedure an improved differentiation was observed. The cells were observed at different time points using an inverted microscope equipped with a 20 × /40 × magnification objective (Leica DMI4000B microscope). Typically, 10 fields per sample were acquired to perform morphometric analysis of PC12 differentiation. Three parameters were measured as previously reported  cells were maintained in a humidified atmosphere at 37°C, 5% CO2 in Primary Neuron Basal Medium  supplemented with L-glutamine, antibiotics and NSF-1  as recommended by the manufacturer. Neurons were plated on the substrates previously coated with a PBS solution of 30 μg/ml Poly-D-lysine (Sigma-Aldrich) (PDL) and 5 μg/ml laminin (Life Technologies). The medium was always supplemented with 100 ng/ml of NGF (Alomone Labs). Since 24 h after seeding, 25 μM AraC (Sigma-Aldrich) was added for inhibition of glia proliferation. Half of the medium was replaced every 3–4 days. Neurons were observed at different time points using an inverted microscope (Leica DMI4000B microscope).*p < 0.05, **p < 0.01, and ***p < 0.001.For all the experiments, we performed two independent cultures with two biological duplicates each. For the morphometric analysis of the PC12 cells, for each substrate we analyzed at least 200 cells (nc = number of cell) from selected fields (nf = number of field) of the four replicates  obtained with a 40 × objective . For the DRG neurons we analyzed nf fields using a 40 × objective for a total of nc cells for each substrate . The number of cells analyzed (nc) is the total pool of the four experiments. All data are expressed as the average value (mean) ± standard error of the mean (SE) unless stated otherwise. Data were analyzed by using Origin Software and nonparametric Kruskal–Wallis test with Dunn's multiple comparison test were used for statistical significance with NGF-induced neurite outgrowth of PC12 cells is favored by their adhesion on a substrate. This is typically achieved by coating the dish surfaces with polymers such as poly-L-lysine or biologically derived collagen  alone and PDL with laminin. Figure Interestingly, the coating solutions distributed differently on graphene and SiC, despite their similar morphologies before the coating, with nanometric terraces and comparable roughness . All polymeric coatings exhibited similar distributions on SiC, while there were significant differences between the coatings on graphene. The dissimilar arrangement of the coatings on the substrates can be reasonably ascribed to the different hydrophilicity of graphene and SiC , well (***) and SiC (*) by 27, 22, and 13%, respectively. The percentage of differentiation on graphene was better than on glass (*), while the average number of neurites per cell was lower on graphene than on the control well (***). These results indicate that PC12 cells grow longer neurites on graphene, with a neuronal differentiation that is comparable to that obtained for the standard control wells. Differently from reference  cells while using the same controls adopted in the previous culture. As motivated in section Polymeric Coating of Epitaxial Graphene and Control Substrates, all the samples were coated with PDL/laminin. Figure Given that neuronal growth was previously reported also for non-coated graphene  at 5 days of differentiation when compared to controls. Remarkably, graphene performs better than gold, which we used as conductive control. Culture of DRG neurons also shows a positive outcome on graphene: neurons survive both on bare and coated graphene until day 17, with a dense axon network that is comparable to the control substrates. In order to investigate graphene influence on axonal outgrowth, further studies are necessary, e.g., using compartmentalized chambers (Taylor et al., DC, LM, and CC conceptualized the study, DC performed the experiments. All the authors discussed the results. The manuscript was written through contribution of all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The breakthrough of next generation sequencing-technologies has enabled large-scale studies of natural microbial communities and the 16S rRNA genes have been widely used as a phylogenetic marker to study community structure. However, major limitations of this approach are that neither strain-level resolution nor genomic context of microorganisms can be provided. This information, however, is crucial to answer fundamental questions about the temporal stability and distinctiveness of natural microbial communities.We developed a methodological framework for metagenomic single nucleotide polymorphism (SNP) variation analysis and applied it to publicly available data from 252 human fecal samples from 207 European and North American individuals. We further analyzed samples from 43 healthy subjects that were sampled at least twice over time intervals of up to one year and measured population similarities of dominant gut species.We detected 10.3 million SNPs in 101 species, which nearly amounts to the number identified in more than 1,000 humans.The most striking result was that host-specific strains appear to be retained over long time periods. This indicates that individual-specific strains are not easily exchanged with the environment and furthermore, that an individuals appear to have a unique metagenomic genotype. This, in turn, is linked to implications for human gut physiology, such as the stability of antibiotic resistance potential.


An inkjet-printed relative humidity sensor based on capacitive changes which responds to different humidity levels in the environment is presented in this work. The inkjet-printed silver interdigitated electrodes configuration on the paper substrate allowed for the fabrication of a functional proof-of-concept of the relative humidity sensor, by using the paper itself as a sensing material. The sensor sensitivity in terms of relative humidity changes was calculated to be around 2 pF/RH %. The response time against different temperature steps from 3 to 85 °C was fairly constant (about 4–5 min), and it was considered fast for the aimed application, a smart label. This is due to its attractive characteristics, such as being a widely available substrate and a low-cost material. Its flexibility allows a wide range of different applications, and the biodegradability is very important when thinking about environmentally-friendly processes and applications. Nevertheless, this paper presents challenges that polymer-based substrates do not present; for example, the porosity or surface roughness of the substrate material, which are crucial parameters when printing conductive lines. However, with polymer-based substrates, one of the crucial parameters is the glass transition temperature  of the surroundings. Usually, continuous and reversible humidity sensing is based on the exposure of thermoset polymers to different relative humidity conditions, followed by reversible dielectric permittivity change . IdeallyThis paper introduces the use of paper not only as flexible substrate material, but also as active sensing layer, responsive to relative humidity changes. It is an advantage over conventional IDE sensors. It requires no additional steps such as coating or passivation layers, and uses commercial paper. The use of fewer layers introduces less errors and faster fabrication process. Inkjet-printing was used to fabricate the conductive electrodes, using commercial Ag ink. Commercial paper was used as substrate and sensing material. It was exposed to changes in relative humidity from 40 to 100 RH %, at different temperatures from 30 and 85 °C, with an almost linear behaviour, small hysteresis, good response time of less than 5 min, and high reproducibility.2) from Stora Enso  was used as substrate material. A commercially available silver (Ag) nanoparticle colloidal ink (ANP 40LT15C) from Advanced Nano Products Co., Ltd.  was used in this work. The metallic nanoparticle-based ink contains around 30 wt % Ag nanoparticles in a polar solvent, with a particle diameter of around 5–10 nm.Lumi silk  to visually inspect the quality of the printed line. A Veeco optical interferometer equipped with a Wyko NT9100 detector  was used to measure the physical parameters of the printed structures  and acquire a topographical image on a 5 mm × 5 mm area, with 5× magnification.Inkjet-printing was carried out using a piezoelectric multi nozzle from Dimatix , with 10 pL cartridges. The drop spacing was about 30 µm. While printing, the substrate temperature was set to 60 °C in order to improve the evaporation of the solvent from the ejected droplets. The ink-jetted structures were printed onto the substrates without any pre-treatment. In the IDE configuration, the line width of the fingers structure  was set to 200 μm, with 150 μm gap and 900 μm length, occupying a total surface area of 1 cm × 2 cm as in Infra-red (IR) was used for sintering the printed devices, using an IR-oven Infrared IC heater T-962 Puhui  with 800 W output power. After the printing of the structures, the substrates were introduced in an IR-oven at 160 °C for 7 min.Changes in capacitance were measured using an HP 4192A LF impedance analyser (5 Hz–13 MHz) at a fixed frequency of 100 kHz every 10 s. The choice of frequency was due to its compatibility with wireless read-out. The devices were chosen and connected to the impedance analyser and were introduced in an SH-221, from ESPEC Corp . humidity chamber with a wet and dry airflow control, at different humidity steps in the range of 40–100 RH % and temperatures in the range of 30 to 85 °C, for a fixed period of 20 min each. The data was collected and recorded through a PC, using in-built software. A commercial sensor Tiny Tag Plus 2, TGP-4500  was used to register and control the exact temperature and humidity levels inside the humidity chamber every 10 s in order to verify the chamber accuracy. Both sensors, printed and commercial, were placed in the middle of the chamber in order to reach the equilibrium and interact in the same way with the surrounding environment.Typically, the conventional fabrication process requires a sensing material, either deposited or printed on top of the IDE. In this work, no such layer was introduced, using the paper as substrate and as sensing material. A piezoelectric inkjet-printer was used to print the patterns in an IDE configuration. This configuration was designed as shown in Optical microscope images were taken of the surface of the IR-sintered lines on the paper substrates, after sintering see .Physical dimensions of the finger electrodes were measured with the optical profilometer, and the printed structures showed average width of 208.0 ± 15.7 µm, a gap between fingers of 140.0 ± 9.4 µm, and average thickness of 185.4 ± 69.4 nm. These values are slightly discrepant from the aimed dimensions due to the wetting of the ink on the paper surface and the paper porosity. Usually, during the process of depositing ink onto the substrate surface of the paper, a part of the ink immediately soaks the paper fibres and penetrates the paper. This not only promotes the drying process, increasing the evaporation rate of the ink solvent, but also improves adhesion. Simultaneously, it improves high print resolution, avoiding spreading on the substrate surface, improving the edge line roughness definition. From In the author’s previous work , a set o0. Relative humidity values come from the wet and dry airflow control.Initial capacitive humidity measurements were made in a humidity chamber at a fixed temperature of 40 °C. The printed sensors were tested under a wide humidity range from 40 to 100 RH %, in 20 min intervals, increasing and decreasing the humidity steps. The humidity levels were set up in this interval due to the physical constraints of the humidity chamber. The selected time period was set up to 20 min in order to allow the diffusion of water vapour molecules to be adsorbed into the substrate and stabilize, reaching the equilibrium with the environment. The output signals of both sensors were normalized to the nominal value of capacitance CGood reproducibility of the printed sensor is shown in This behaviour shows hysteresis of the printed sensor in an up and down cycle. This might be due to saturation of the paper substrate sensing material. The explanation for that is quite simple and it is due to the adsorption and desorption process for an IDE sensor, when exposed to different relative humidity levels. When the relative humidity is low (below 40 RH %), the sensing material will uptake and adsorb the water vapour molecules, usually swelling rapidly. If the relative humidity increases to higher levels , the sensing material will swell more, adding more water vapour molecules adsorbed to the previous ones. After that, if the relative humidity is reduced to the initial level (40 RH %), the sensing material will take more time to desorb and to reach the equilibrium with the surroundings. That usually translates into a slow de-swelling, due to saturation. The transition between the swell and de-swell steps depends on the sensitivity of the material towards the uptake of water vapour molecules and the ability to desorb them and reach equilibrium. In the case of paper substrate, resuming the adsorption process might be relatively fast, whereas desorption and the process to reach equilibrium state will be slower, due to the soaking of the fibres in paper.Another way to represent the UP and DOWN cycles—or the hysteresis of the inkjet-printed sensor—is represented in These UP and DOWN cycles refer to an increase in relative humidity (UP) and a decrease in relative humidity (DOWN) for the entire measurement cycle. The coefficient of linearity is 0.90 and 0.96, as calculated for the UP and DOWN cycles, respectively. It is then inferred that the inkjet-printed sensors have an almost linear response against humidity, which translates into a small hysteresis loop after long-term operation. This means that the response is very similar in different relative humidity levels. Hysteresis is a typical and significant problem in capacitive sensors. As explained before, it derives from the absorbed water inside the paper structure, increasing the swelling of the fibres, making it slower to desorb and reach the equilibrium, causing a variation in the linearity of the sensor, and thus, hysteresis. It can be improved by reducing the paper thickness, because if the adsorption layer is thinner , it willThe response time of the IDE inkjet-printed sensors on paper substrate was calculated for humidity steps from 40 to 100 RH %, as represented in The response time of the IDE printed sensor was about 5 min, whereas the recovery time of the IDE printed sensor was less than 4 min for about 60 RH %. For high humidity levels of 100 RH %, the response time of the IDE printed sensor was about 4 min, whereas the recovery time of the IDE printed sensor was less than 3 min. Although the printed sensors cannot compete with the commercial humidity sensors, with response times between 4 s and 47 s ,32,33, oThe effect of the different temperatures on the relative humidity response and on the repeatability of the sensor performance was also studied with the inkjet-printed sensors, with a minimum of three parallel samples. Temperature is one of the most important parameters that interferes with the response of the inkjet-printed sensors.For lower humidity rates (around 40 RH %), the sensors show small sensitivity to temperature changes, whereas above this humidity level, the sensors show higher sensitivity. The temperature effect takes place at 60 °C and remains somewhat the same up to 85 °C, being more pronounced at higher humidity rates . Above 85 °C, the sensors show some instability, probably due to degradation of the substrate and/or the printed layer. The temperature sensitivity of the inkjet-printed sensors can become negligible when applied to low-cost, mass-manufactured environmental sensors, as referred to previously. Nevertheless, the achieved performance was considered acceptable for inkjet-printed moisture sensors on paper substrate when compared with traditional commercial sensor and other printed sensors reported in the literature, as is summarized in Courbat et al.  developeTo improve the sensor sensibility and simultaneously reduce the oxidation of Ag, a well-known phenomenon due to exposure to humidity, electrodeposition of a thin metal layer can be performed. This will be addressed in future works as a way to reduce the influence of humidity and temperature on the sensor response. The electrodeposition of a thin film of nickel and copper as a way to passivate the inkjet-printed lines has been described in previous works ,35. ThisAll-inkjet-printed capacitive-type humidity sensors on a paper substrate which is sensitive to moisture were demonstrated herein. Inkjet-printing was shown to be a convenient, easy, fast, and cheap way to produce IDE configuration relative humidity sensors on flexible substrates such as paper with a good printing quality, resolution, and high fabrication yield. Inkjet-printing has the advantage, as a sheet-to-sheet (S2S) production technique, to be up-scalable to roll-to-roll (R2R) mass manufacturing, thus reducing drastically the productions costs. The realized inkjet-printed paper sensors showed to be comparable to commercial sensors ,32,33 in


Transport of coral reef fish larvae is driven by advection in ocean currents and larval swimming. However, for swimming to be advantageous, larvae must use external stimuli as guides. One potential stimulus is “odor” emanating from settlement sites , signaling the upstream location of desirable settlement habitat. However, specific chemicals used by fish larvae have not been identified. Dimethyl sulfide (DMS) is produced in large quantities at coral reefs and may be important in larval orientation. In this study, a choice-chamber (shuttle box) was used to assess preference of 28 pre-settlement stage larvae from reef fish species for seawater with DMS. Swimming behavior was examined by video-tracking of larval swimming patterns in control and DMS seawater. We found common responses to DMS across reef fish taxa - a preference for water with DMS and change in swimming behavior - reflecting a switch to “exploratory behavior”. An open water species displayed no response to DMS. Affinity for and swimming response to DMS would allow a fish larva to locate its source and enhance its ability to find settlement habitat. Moreover, it may help them locate prey accumulating in fronts, eddies, and thin layers, where DMS is also produced. As the larva develops it swims in an active2 and directed3 manner, utilizing its impressive swimming and sensory abilities4. Furthermore, biophysical models of larval transport show that the behaviorally-mediated swimming during this stage increases local retention and enhances overall settlement success6. As a result, studying the complex behavioral responses of larvae to their sensory environment has emerged as a major component of understanding marine dispersal and population connectivity4, focusing on chemical11, auditory13, and celestial15 stimuli.The early life history of most reef fishes is a multi-stage process during which they are capable of long-distance dispersal; however, the behaviors influencing dispersal are poorly understood. Immediately post-hatch, a larva possesses weak sensory capabilities and limited mobility, thus its transport is presumably passive17. It is not always clear whether olfaction  or gustation  is at work, but both chemosensory systems are well developed in fishes18. There is a long history of research on settlement-inducing chemoreception in marine invertebrates19, as well as on chemically-induced settlement in fish larvae20. Several marine fishes have even evolved the capacity to utilize distinct chemical signals at impressively large spatial scales, specifically salmon22 and sharks26. Nonetheless, little is known about what chemicals play an important role, how perception of these chemicals affects swimming patterns, or mechanisms for their effective use in navigation27.There is strong physiological and evolutionary evidence to suggest that olfaction is the most primitive and universal sense, and that it is intricately linked to odor-tracking and the use of a cognitive odor map7. Population genetic substructure provides additional support for this hypothesis28, as does the activation of oriented swimming when larvae enter such a tidal plume10. Also, like salmon, some coral reef fish appear capable of imprinting as early as embryonic development and into the first 24 hours post-hatch29. The expression of this imprinting seems to vary throughout ontogeny, with chemical preference sharply transitioning from that of offshore waters to that of settlement habitat in one case30. It seems very clear that settlement-stage larvae of many reef fishes can and do discriminate chemical environments, preferring those of appropriate settlement habitat and of conspecifics32. However, despite this breadth of literature, there is only a weak understanding of exactly what chemicals appeal to larvae .The literature suggesting that larval fish also utilize chemosensory systems for large scale navigation continues to grow. Researchers have hypothesized that “tidal halos” extending several kilometers from settlement sites, which are rich in chemical compounds carried from the reef, may provide important cues for navigation and settlement at varying spatial scales33. Since then it has gained attention in the biological community for its role as a chemical attractant34. And while most of this work has focused on marine air-breathers such as seabirds36, penguins37, turtles38, and seals39, recent work has also demonstrated an affinity in whale sharks40. Dimethylsulfoniopropionate (DMSP), a precursor to DMS, has been shown to be a foraging cue for adult reef fishes41. Since many larval fishes have highly-developed olfactory systems even before juvenile metamorphosis42, it is reasonable to assume that larval fish would have an affinity for similar chemicals.A logical candidate for a specific chemical involved in larval fish navigation is dimethyl sulfide (DMS), a particularly well-known “infochemical” in the marine environment. DMS has been well studied in the scientific community as far back as 1980’s for its potential role in climate regulation43, corals44, benthic algae45, and other coral reef residents. Surface concentrations of DMS(P) are higher near certain coral reefs than anywhere else in the marine environment (as high as nearly 19 μM [DMS] in one area), particularly in the mucus ropes and surface films exported from the reef as a part of the tidal halo46. Midwater samples of DMS(P) vary annually, but average from 20 nM to 120 nM47, with events such as coral spawning leading to temporal spikes in background concentrations. One further important consideration is that all methods for collecting water samples necessarily lead to an averaging of the DMS(P) concentration over the volume collected. In a chemically heterogeneous environment, chemical concentrations may be much higher at finer spatial scales, and small planktonic organisms are likely to perceive these higher concentrations48. In any case, it is clear that, despite considerable temporal and between-reef variation, concentrations are consistently higher around corals than in the pelagic environment. Thus, one would expect that an ability to detect and an affinity for DMS(P) would enhance the recruitment of larval fish at their reef destinations.Moreover, high concentrations of DMS(P) are produced by zooxanthellaeCoryphaena hippurus, a tropical epipelagic fish that spends all its life cycle in the open ocean.In the present study, we utilize a shuttle box (Loligo Systems) adapted to serve as a choice chamber, with DMS-containing seawater on one side and control seawater on the other. We hypothesized that the reef fish larvae would display a preference for the water containing DMS and, further, that their swimming behavior would be altered when they are inside of this water. Experiments were conducted on a range of different reef fish families to determine if observations were consistent for fish larvae with varying ecological traits. Further, in order to assess the relative importance of DMS in indicating reef habitat or food abundance, experiments were also conducted with larvae of mahi-mahi, Stegastes spp larvae  displayed preference for the side containing DMS, including 16 of the 19 (84.2%) % displayThe normalized count-difference variable was used to statistically asses the group-level preference for DMS odor. Figure There were also clear differences in behavior of reef fish larvae when in water containing DMS odor. First, we found differences in the activity level  of larvae while in odorous water  displayed preference for the side containing DMS. Pooling all trials, including those with a control-side preference, larvae spent 56.2% of their time on the odor side, 1.3x higher than the 42.7% spent on the control side. The remaining 1.1% was spent inside of the center pathway.The normalized count-difference variable was used to statistically asses the group-level preference for DMS odor. Figure et al.10 when larvae were inside of tidal halos. We did not, however, observe any preference for DMS in larvae of the epipelagic mahi-mahi, nor did we observe any change in behavior.We demonstrate that reef fish larvae, across taxa, display a preference for seawater containing DMS. Furthermore, the presence of DMS in the water elicits a different swimming behavior than water without DMS, causing them to swim slower and make larger turns more frequently, consistent with observations in Paris 28 or in showing that a single species prefers a particular “leachate”9, but this is the first example of cross-taxon reef fish preference for a specific isolated compound. Over the past decade there is an impressive amount of literature displaying the potency of DMS as a universal marine infochemical41, and this study marks the addition of reef fish larvae to the growing list of attracted organisms.The conclusion that reef fish larvae generally possess an affinity for DMS is a significant one. Previous studies have been successful in demonstrating a preference for a chemical suite28. The primary benefit of this new method is the ability to simultaneously test the appeal of (or aversion to) a chemical and its effect on the movement patterns of a larva. In this way we are able to assess response to the odor in addition to preference for it. There are, however, a few important remarks regarding the use of a shuttle box for this application: (1) often larvae spend a considerable amount of time in the center pathway connecting the control seawater and the DMS containing seawater. This is likely due to the reduced flow in this region, something which, under stress, may be more important than chemosensory appeal; (2) certain larvae are unsuitable for testing with the shuttle box. Some Ostraciidae and Tetraodontidae larvae, for example, simply did not move. These larvae would stay on the side of the shuttle box they were placed in until the experiment ended, regardless of the length of the experiment. Similar behavior has been observed in subadult Lutjanus griseus inside of a shuttle box designed to investigate salinity preference51. Some Gerreidae and Clupeidae larvae, on the other hand, would energetically swim into the nearest wall as soon as they were placed into the shuttle box. This places a restriction on which taxa one can reasonably test using this device. We suggest this is also the case for other methods investigating chemical preference in larvae, based on personal communications, and that this is generally underreported. (3) Nearly all larvae spent some time in the control water, despite their significant preference for DMS water. This is likely a result of odor-induced search behavior, whereby the larvae were incited to find the source.This study also marks the first use of a shuttle box to assess chemical preference of an aquatic organism. Typically, preference experiments with larval fish have utilized a two-channel flume34. Specific to fish larvae, high concentrations of DMS also suggest the nearby presence of suitable settlement habitat. It is unclear which of these two functions facilitated the evolution of DMS-preference, but it is noteworthy that the concentrations of DMS are higher in coral reef exports  than anywhere else in the marine environment, and that the background concentrations are particularly low elsewhere in the tropics46. For example, mucus ropes may contain anywhere from 60 to over 12000 times higher DMS concentrations (132 nM–17909 μM) than background (2.2 nM), suggesting DMS may be more useful in finding the reef than in finding prey. Our results here are also telling. While reef fish larvae displayed a strong preference for DMS, mahi mahi did not. This is not surprising, as DMS levels are substantially lower in the epipelagic environment than near reefs.Like other organisms with DMS-affinity, fish larvae may use the presence of DMS as an indicator for nearby food sources41. It is unclear whether DMS or DMSP is the “more potent attractant”, if they serve different roles, or if the ratio of the two chemicals is in some way important information for early or late stage larvae.However, it is also possible that DMS actually serves both roles, either simultaneously or at different points in development of the larvae through its pelagic duration. Indeed, DMS is actually a product of the degradation of DMSP, a chemical which several species of adult fish have a documented preference for29 and behavioral52, that supports the idea of chemical imprinting early in fish development, and this study does not intend to refute those findings. Indeed, imprinting on specific microhabitat odors likely plays a very important role in the selection of settlement habitat at a local scale. We speculate that DMS, which is more ubiquitously present at a reef, and would be more broadly exported from it, may be more important as a large-scale infochemical. However, we point out that damselfish of the Stegastes genus made up the bulk of our sample, demonstrating that benthic spawners, those most likely to imprint, still display an affinity for DMS. It is possible that DMS, despite its ubiquitous presence on the reef, is actually implicated in the process of imprinting, either in isolation or as one of a suite of chemicals.The idea of a widely-appealing chemical is relatively new and needs to be discussed in the context of perhaps the longest-standing hypothesis in the chemical ecology of larval fish: the imprinting hypothesis. There is strong evidence, both physiological53. The structure depends on the dominant flow, turbulent diffusion, topography, etc.48 and creates a serious problem for traditional gradient-based odor-tracking strategies. Knowing the threshold of detection for DMS will allow observations of behavior to be compared to models of behavioral responses.This study is an important step forward in understanding the chemosensory navigation of fish larvae and allows us to confidently pursue additional questions. For example: (1) what is the threshold of detection for DMS? or (2) what is the mechanism for navigating using odor plumes? Clear evidence of chemical preferences certainly suggests a mechanism exists, but its nature is unknown. The spatial and temporal distributions of chemical concentrations are notoriously complex and composed of highly-concentrated filaments interspersed with fluid containing little or no chemicalWhich strategies are being used by minute fish larvae to find their settlement sites from the open ocean remains a mystery. Future studies should use DMS, at relevant concentrations above threshold of detection, to simultaneously record odor plume structure and larval fish kinematics. This will provide valuable insight on the mechanism being used to find the source of the odor. The identification of DMS as a universal infochemical for larval reef fish brings us closer to realizing this goal.54 and incidental mortalities allowed certain specimens to be saved for detailed taxonomy. All other individuals were freed in the nearby bay following their behavioral trial, in accordance with the guidelines of University of Miami IACUC protocol #14-216. All methods were carried out in accordance with relevant guidelines and regulations, defined in said protocol, approved by University of Miami’s Institutional Animal Care and Use Committee. A total of 28 larvae belonging to 10 taxa were caught and used in experiments,  and Stegastes diencaeus (n = 7). All larvae were wild-caught opportunistically from light-traps, and thus experiments were conducted on a wide array of fish families. Only one genus of damselfish, Stegastes, had substantial representation in our dataset (n = 17).Larval fish used in the experiment were obtained by one of two ways: 1) Wild-caught utilizing light traps, or (2) Reared in the University of Miami Experimental Hatchery (UMEH) at the Rosenstiel School of Marine and Atmospheric Science. Light traps  were set on fore reef off the coast of Key Biscayne, FL, USA . Traps were set at dusk and fish were retrieved from traps at dawn. All experiments were conducted within 24 hours of larval collection, and all larvae were drip-acclimated to the water from the seawater system in the Marine Technology & Life Sciences Seawater Research Building at University of Miami’s Rosenstiel School. Identifications were made using morphological key characteristics Wild-cauet al.55 and Kraul (1993)56 for rearing of cobia (Rachycentron canadum) and mahi-mahi, respectively. Eggs were hatched and larvae were reared in 2.4 m3 cylindrical fiberglass tanks. Beginning on day 3 post hatch the rearing water was inoculated with Nannochloropsis microalgae paste (Reed Mariculture Inc.) at a level to maintain a slight green color in the water column . Enriched rotifers (Briachionus plicatilis) and Artemia nauplii were fed to the larvae over the course of the rearing period, and water exchange averaged 10 turnovers per day in the rearing tanks. Larvae used for this study were randomly removed from the larval rearing tanks using a submerged 1-L beaker to avoid physical contact and they were subsequently held in 15-L vessels containing UV-sterilized aerated seawater in the hours leading up to testing.Mahi-mahi larvae were reared from fertilized eggs obtained from volitional spawning wild mahi-mahi broodstock maintained at the UMEH facility. Larval rearing protocols were similar to those described by Benetti A novel methodology utilizing a shuttle box design . Next, the shuttle box was filled with approximately 175 mL of water in each chamber and a larva was randomly introduced to one side. The experiment began when the pump was activated, introducing seawater with 340 nM DMS on side and seawater with 0 nM DMS on the other; sides were chosen at random. 340 nM was chosen as the stock concentration based on the maximum measurement of DMS in sediment pore waters at Kelso Reef, a mid-shelf oligotrophic patch reef in the central Great Barrier Reef46. No one was permitted to enter the experimental room during these 20 minutes, and the movement of the larva inside of the shuttle box was recorded by a video camera set to 30 frames per second. At the end of the trial the larva was removed, the shuttle box and tubing was thoroughly rinsed with ethanol and seawater, and the sides for larval introduction and odor were randomly chosen for the next experiment.The 20 minutes of each trial was split into 5 minutes of acclimation and 15 minutes of experiment. The acclimation period allowed the larvae to adjust to their new environment and also for the concentration of DMS to accumulate inside one of the chambers  to record the position of the larva inside the shuttle box at 1 second intervals. Custom software was designed integrating R  and ImageJ  to process each image sequence. Briefly, the user manually selected the boundary of both chambers, used a portion of the shuttle box with known length to calibrate pixels to centimeters, and then selected the approximate position of the larva’s head in every frame. The program used these positional records to determine the amount of time spent in each chamber, instantaneous velocities, and turning angles. Records of larval position in the center pathway were excluded from analysis.Video data was subsampled using VirtualDub  was used to test the hypothesis that the mean was indeed equal to 0 when data met the assumptions of normality, independence, and random sampling. For data that was not normally distributed, a nonparametric Wilcoxon signed rank test was used. Because positional records where the larvae is in the center pathway were excluded, this variable was normalized to account for the different number of records between trials . This was accomplished by removing those records from the dataset and dividing the count difference by the number of records remaining.In addition to side preference, behavioral differences while in each side of the chamber were assessed. Specifically, the mean swimming speed  and the variation in swimming speed were compared. The frequency of large turns  was also compared. This latter variable was normalized to account for the fact that larva may have spent more time on one side than another, increasing the frequency of turns on that side without indicating a change in behavior. This was accomplished by dividing the turning frequency on each side by the total number of records on that side. A paired t-test (α = 0.01) was used to test the hypothesis that mean turning frequencies and activity levels were equal in seawater containing DMS and control seawater when our data met assumptions of normality, independence, and equal variance between groups, otherwise a nonparametric paired Wilcoxon signed rank test was used. Finally, while tracking was done without knowledge of the odorous side, a qualitative assessment of swimming behavior on each side was used to explain quantitative findings.Supplementary InformationSupplementary Video


In this study, different types of graphene, including single-, few-, and multi-layer graphene, were grown on a stainless steel (SS) mesh coated with Cu catalyst by using the chemical vapor deposition (CVD) method. Even though the SS mesh consisted of different types of metals, such as Fe, Ni, and Cr, which can also be used as catalysts, the reason for coating Cu catalyst on the SS surface had been related to the nature of the Cu, which promotes the growth of graphene with high quality and quantity at low temperature and time. The reaction temperature and run time, as the most important parameters of the CVD method, were varied, and thus led to the synthesis of different layers of graphene. Moreover, the presence of single-, few-, and multi-layer graphene was confirmed by employing two techniques, namely transmission electron microscopy (TEM) and Raman spectroscopy. On top of that, electron dispersive X-ray (EDX) was further applied to establish the influence of the CVD parameters on the growth of graphene. This nanomaterial, a two-dimensional 2D) material, is formed by spoperties , 2. In fD materiaoperties –6. Graphoperties , 8.Since decades ago, coating carbonic materials on steel in the steel industry has been carried out to improve the steel properties by using of a low amount of carbon materials . Among tIn this process, steel is placed into a carbon-rich environment at high temperatures for a certain amount of time and then quenched. Although the processes for coating the metal with carbonic material are different from the rest, the chemical vapor deposition (CVD) technique is generally used to grow graphene on the surface of the metals. Therefore, CVD, as one of the most frequently used techniques, was applied to synthesize graphene , 14. FurIn addition, to achieve the different layers of graphene  on stainless steel substrates, some parameters of the CVD method should be optimized. Therefore, the most important parameters of the CVD technique, such as reaction temperature and time, which affected the graphitization, the structures, and the amount of the different layers of graphene on the SS surface, had been changed. Hence, by controlling these two parameters, the number of graphene layers changed as well. Besides, the SS surface was coated with Cu solution, which had never been attempted before, to promote high quality of graphene sheet on SS surface. On top of that, some analytical instruments were applied to evaluate the resulting graphene. Raman spectroscopy, as a non-invasive method, was utilized to determine the number of graphene layers and graphitization . Other t3)2.3H2O solution and sonicated for 2 h to coat the surface of SS with Cu particles. The EDX results of SS mesh coated with Cu are as follows: C 0%, O 5.3%, Si 0.21%, Cr 10.2%, Ni 5.2%, Fe 46.2%, and Cu 32.2%, while the remaining weight was related to other components. The presence of oxygen was associated with the elemental composition of Cu nitrate trihydrate. Besides, when the SS mesh was placed into the CVD reactor and the temperature was increased to 300 °C, the nitrate compound was removed.In this work, stainless steel  that comprised of 50-μm diameter fibers was purchased and applied as a substrate to synthesize the graphitic structures on its surface. The composition of the SS mesh was obtained by using EDX and is given in the following: C 0 wt%, Si 0.53 wt%, Cr 19.10 wt%, Ni 8.6 wt%, and Fe 70.3 wt%, while the rest had been related to other components. The pristine SS mesh was cut into a circle shape with 2-cm diameter and immersed into 9 M sulfuric acid solution for a few minutes to pretreat the SS surface. After that, the SS mesh was immersed into 50 mM Cu(NO2H2) was used as the hydrocarbon source, while nitrogen (N2) and hydrogen (H2) were utilized as the carrier gases. Next, the reaction temperature and the time in the CVD technique were altered to grow the graphene with different layers. First, the SS mesh was located in the CVD chamber, and then, the temperature was increased  under the flow of N2 gas. When the system reached the required temperature, the acetylene gas as a carbon source was inserted into the system at 50 sccm with the presence of H2/N2 flow (10 ssm/50 sccm), for different run times from 10 to 50 min. Finally, the C2H2 flow rate was stopped, and the furnace was turned off under the presence of N2 flow. Thus, in order to evaluate the morphology, graphitization and composition of the produced nanomaterials, Raman spectroscopy, SEM, TEM, and EDX were employed.In order to synthesize the graphene, pure acetylene  and 2D peak (~2650 cm−1), in relation to the electronic and geometrical structures through the double-resonance process, were investigated. The D peak provided information about the breathing modes of the sp2 atom [−1), which was associated with an E2g stretching mode of the graphitic structure. The stretching of the C–C bond in graphitic materials gave rise to the G peak, which was related to all sp2 carbon systems. Moreover, the intensity ratio of the D peak and the G peak (ID/IG) was utilized to evaluate the degree of graphitization [2D/IG ratio revealed the high quality and also the low number of graphene layers [2D/IG ratio, the number of graphene layers decreased. The Raman spectra at different temperatures are displayed in Fig. D/IG decreased, but I2D/IG increased, which showed that the graphitization grew with the decrease in the number of graphene layers (increased 2D peak and decreased D peak).To study the influence of the reaction temperature on the structure and the morphology of the products, the temperature was increased from 950 to 1050 °C at 30 min run time under 50/50/10 sccm with Cfast one . In the sp2 atom  activatesp2 atom ). Anothetization . Besidese layers . TherefoMoreover, the TEM images in Fig. 2/H2/C2H2 flow rates for 50/10/50 sccm) to determine the effects of the reaction time on the graphitization, as well as the morphology of the products.Reaction time leads to the activation of the metallic catalyst to grow the different number of layers of graphene with high quality and graphitization. Therefore, the time had to be optimized for each type of graphitic structures by using different times  while the other parameters were kept constant (at 1050 °C under N2D/IG ratio was about 2) [Based on the Raman spectroscopy, it was found that by changing the reaction time, the intensity of the 2D and D peaks of the Raman spectra altered significantly Fig. . So, by about 2) , which wD/IG = 0.4 and I2D/IG = 1.94). Meanwhile, the FLG with 24.8 wt.% C atoms was grown at 1050 °C for 30 min under the 50 sccm acetylene flow rate with high graphitization (ID/IG = 0.1 and I2D/IG = 0.75). Other than that, the MLG with about 22.6 wt.% C atoms was synthesized at 1000 °C for 30 min under the 50 sccm acetylene flow rate with low graphitization (ID/IG = 0.5 and I2D/IG = 0.45). Furthermore, by increasing the temperature to 1050 °C for 50 min, the MLG was achieved with 27.8 wt.% C atoms and the highest graphitization was attained (ID/IG = 0.05 and I2D/IG = 0.5). Nonetheless, it is worth noting that the growing of the structures with low graphitization at 950 °C for 30 min under the 50 sccm was related to non-graphene materials (ID/IG = 0.95 and I2D/IG = 0.24).The most important goal of this research was to grow different layers of graphene on the SS mesh. Hence, a simple method was employed to fabricate different layers of graphene sheets on SS surface coated with Cu particles as a promoting catalyst for graphene growth in a large scale with high quality via CVD technique. Therefore, in order to determine the different layers of graphene, some parameters of the CVD process had been changed. The reaction temperature was ranged from 950 to 1050 °C, while the reaction time was varied from 10 to 50 min. On the other hand, the effects of these parameters on the graphitization, the number of graphene layers, and the amount of C atoms of the product were demonstrated by Raman spectroscopy and EDX, respectively. In addition, SEM and TEM were used to analyze the morphology and the structures of the graphene on SS mesh. Based on the results, single-layer graphene on SS with about 10.2 wt.% C atoms was grown at 1050 °C for 10 min under the 50 sccm acetylene flow rate with low graphitization (I