Policing duties may inherently be dangerous due to stab, blunt trauma and ballistic threats. The addition of individual light armor vests (ILAVs) has been suggested as a means to protect officers. However, the addition of the extra load of the ILAV may affect officer ability to conduct occupational tasks. The purpose of this study was to determine if wearing any of three different ILAVs made by different companies with their preferred materials and designs  affected occupational task performance when compared to that in normal station wear. A prospective, within-subjects repeated measures design was employed, using a counterbalanced randomization in which each ILAV was worn for an entire day while officers completed a variety of occupationally relevant tasks. These tasks included a victim drag, car exit and 5-meter sprint, step down and marksmanship task. To compare the effects of the ILAVs on these tasks, a multivariate repeated measures analysis of variance (ANOVA) was conducted, with post hoc pairwise comparisons using a Bonferroni adjustment. Results showed that performance in each task did not vary between any of the ILAV or normal station wear conditions. There was less variability in the marksmanship task with ILAV B, however. The results suggest that none of the ILAVs used in this study were heavy enough to significantly affect task performance in the assessed tasks when compared to wearing normal station wear.  In an age of increasing threats, police forces are more commonly utilizing Individual Light Armor Vests (ILAVs) for protection of their officers from occupational risks such as stabbing, blunt trauma, and gunshot wounds ,2. The IThere have been numerous investigations into the negative effects of carrying excessive loads in military populations ,5,6,7,8,Prior to any decisions being made on the large-scale recommendation and utilization of ILAVs by police forces, a greater understanding is required of both the effects of the added ILAV loads, and any potential differences in occupational performance resulting from the wearing of specific types of ILAV. Therefore, the purpose of this study was to compare the effects of wearing each of three different ILAVs and of normal station wear on the performance of occupational tasks in police officers. To ensure the sample selected was representative of the general state police population, two small-, medium- and larger-stature male and female serving police officers were initially recruited. This process was designed to ensure the translatability of this research to the entire police force and to provide understanding of the effects of the ILAVs when applied to a range of body sizes from both sexes. Each officer was initially briefed, and all expressed a willingness to participate, providing written informed consent. One female officer was removed at this stage from the study due to a medical concern, and the sample was reduced to 11 officers, comprising five females  and six males .A prospective, within-subjects, repeated measures, study design was employed, using a counterbalanced randomization protocol by which to allocate one of four load condition types, being ILAV types A, B and C and “normal” (N) station wear, where officers wore their own station wear. Each officer served as their own control and, regardless of which load condition they were randomly allocated on the first day of data collection, they progressed to the next load condition type in the following predetermined order: ILAV A, ILAV B, ILAV C, N and from N to ILAV A. Officers were required to wear each of their allocated load conditions for an entire day over the 4 days of the study period. This procedure was aimed at reducing any learning effect and the effect of any external factors such as variable weather conditions, which may have occurred over the duration of the study. Data collection for the study took place at a state police college in 2016 over the 4-day period. The ambient temperature and relative humidity across the testing times ranged from 12 to 24 °C and from 36 to 93 percent, respectively, giving a heat stress index varying between 11.4 and 22.6 °C during the testing period. This study was approved by the Bond University Human Research Ethics Committee (protocol number 15803), and all participants formally consented to participate. Departmental approvals for the conduct of the study and release of this paper were also obtained. The same testing procedure was used each day to minimize diurnal variation. As this data capture was part of a larger study, the measures relevant to this study are displayed in A victim drag scenario was set up utilizing a 60 kg mannequin fitted with a 10 kg weighted vest. The recovery course required the officer to drag the mannequin 6 m directly backward, then negotiate a 90-degree left hand turn through a doorway, before dragging the victim another 4 m to the end of the track . This coA standard police patrol car  was parked on the side of a track for a vehicle exit scenario. Officers were seated in the driver’s seat of the vehicle without a seatbelt on and with both hands on the steering wheel. A verbal command was given by the researcher to start the scenario, whilst their hand was used to break the light beam of the SMARTSPEED timing gate system  and so to start the timer. The officer exited the driver’s side of the vehicle and ran 5 m rearward, with the distance measured using a digital mini-measuring wheel , through the corresponding timing gate as quickly as possible . OfficerThe curb step down task required the officer to step off a 20 cm step onto the Fitness Technology Force Platform to determine the peak ground reaction force. The officers were given a verbal command to ‘go’ and were required to step off the step onto the platform and then off the platform in a natural gait pattern. The aim of this task was to gain an appreciation of the loads placed through the officer’s body when simulating a step off a street curb side.The marksmanship task involved officers engaging a target (human silhouette live fire target with four scoring zones) with a Glock model 22 pistol, firing 26 rounds in total, and was scored over three separate sequences. These three sequences were: Point/proximity shooting (nine rounds); Immediate distance/kneeling (five rounds); and Transition drills/reloading (12 rounds). Each sequence assessed a single or related multiple skill set and these were deemed mandatory and necessary skills for the operational policing environment. To score points for assessable hits, one point was awarded when the whole of the round impression/cut was wholly within the human silhouette. However, if the round cut a line and any portion of the ‘cut’ lay within the next zone, the shooter was awarded the higher points. The maximum score was 104 points. For the duration of the marksmanship task, the volunteer officers were under the authority of the host Police Force and their qualified range instructors. All recorded data, except for data relating to ambient weather conditions, were entered into a data spreadsheet in SPSS version 23 . Initial descriptive analyses were then conducted to provide counts, means, standard deviations and ranges for the included variables, as relevant depending on levels of measurement. These descriptive statistics were derived for each sex and for each body armor type where relevant, as well as for the entire sample. Following these descriptive analyses, multivariate repeated measures analyses of variance (ANOVA) were conducted to examine the effects of body armor type on key performance measures, with post hoc pairwise comparisons using a Bonferroni adjustment. Results were graphed where this approach provided useful visualization of key differences. The overall level of significance was set a priori at 0.05.Data relating to ambient weather conditions were analyzed descriptively to determine the range of ambient temperatures, levels of relative humidity and range of heat stress index scores observed during data collection times on the four data collection days. These have been reported in the Methods section of this report.p < 0.05 for all on Bonferroni post-hoc tests; p < 0.002 for all on Bonferroni post-hoc tests).An overview of the weight of each ILAV can be found in p = 0.529). The results for the victim drag, car exit and step-down task are provided in p = 0.761). The quickest time was recorded with ILAV C, and was 0.09 s (2.6 percent) faster than for ILAV A. Again, however, there were no significant differences between any of the ILAV and N conditions in time to complete the car exit and 5m sprint to cover  = 0.390, p = 0.417). The highest peak force occurring during the step-down task was seen for ILAV B, which was 7.8 percent higher than the lowest peak force, which was associated with ILAV C. There were, however, no significant differences in the peak force readings from the curb step-down task between any of the ILAV and N conditions  = 0.865, p = 0.118). Considering this, as shown in The results of the marksmanship task are shown in The purpose of this study was to determine the effects various ILAV systems had on the occupational task performance of state police officers. In contrast to the effects on military tasks, the addition of the ILAV to normal station wear did not affect officer performance in any of the occupational tasks observed in this study. It is suggested that the weights of the ILAVs and associated equipment loads used by the officers in the current study were not heavy enough to significantly affect performance of the occupational tasks assessed, in contrast to wearing heavier external loads, for example over 20 kg for specialist police  and overp < 0.001) longer in the loaded condition (+0.28 s). It should be noted, however, that in the studies by Carlton et al. [Performance in the victim drag task in this study was not significantly affected by wearing any of the individual ILAV or N load conditions. In contrast, in another study, a variation of this same task, a 10 m victim drag of a mannequin, was shown to be performed significantly more slowly when wearing body armor and carrying additional load of over 20 kg in weight . The lign et al.  and Dempn et al. , the difn et al.  and DempThe step-down task did not differ in peak force readings, regardless of load condition. Previous research on police officers has found a drop landing from a 0.75 m platform while wearing body armor and accessories (7.65 ± 0.73 kg) led to significantly greater peak ground reaction forces upon landing when compared to those observed in an unloaded condition [The results from the marksmanship task showed less variability in shoot scores when wearing ILAV B, but no significant difference overall between any of the ILAV and N conditions. Previous research has shown that marksmanship accuracy can be affected after a load carriage activity for 45 min in soldiers (30.5 ± 1.5 kg)  and afteIt should be acknowledged that these results are reflective of only the observed tasks, and did not extend to other factors that may be important to policing. Despite no observed significant effects of the ILAVs on officer performance of any of the occupational tasks investigated in this study, other considerations which may affect the choice of one ILAV over another may include the subjective opinion of officers, and the effects of the ILAV on mobility and balance and range of motion. Due to the time constraints on this study, there was also no capacity to examine the chronic impacts of sustained ILAV loads on the musculoskeletal system of the officers, and prospective studies designed to examine this issue would be of great value given that officers are increasingly wearing ILAV in their day-to-day duties and for increasing numbers of hours—in some cases constantly during working hours. The results from this study suggest that while ILAVs may be significantly heavier than normal station wear, they may not have a notable impact on police officer performance of victim drag tasks, vehicle exits and marksmanship tasks. In addition, musculoskeletal loadings may not be significantly greater when stepping off a low curb when wearing ILAVs than when wearing normal station wear. Police forces can be confident that the addition of load through adopting ILAVs of similar weights to those investigated in the current study will not significantly affect officer performance of a range of representative occupational tasks. However, a caution is noted in that the chronic musculoskeletal effects of sustained increases in carried loads, even slight increases, require further investigation.


In this paper, one-dimensional (1D) and two-dimensional (2D) graphene-based plasmonic photonic crystals (PhCs) are proposed. The band structures and density of states (DOS) have been numerically investigated. Photonic band gaps (PBGs) are found in both 1D and 2D PhCs. Meanwhile, graphene-based plasmonic PhC nanocavity with resonant frequency around 175 THz, is realized by introducing point defect, where the chemical potential is from 0.085 to 0.25 eV, in a 2D PhC. Also, the bending wvaguide and the beam splitter are realized by introducing the line defect into the 2D PhC. Photonic crystals (PhCs), first proposed by Yablonovitch  and JohnGraphene, a two-dimensional (2D) carbon atomic crystal arranged in a honeycomb lattice , has demOne of the typical structures of graphene-based one-dimensional (1D) plasmonic PhCs is graphene micro-ribbon arrays. Early in 2011, Ju et al. reported the tunability of plasmon excitations and light-plasmon coupling at terahertz in graphene micro-ribbon arrays . Then, NIn this paper, we proposed graphene-based plasmonic PhCs with ultra-small size. Both the band structure and the DOS of 1D and 2D PhCs are numerically studied by using the finite element method (FEM) with Comsol Multiphysics Radio Frequency (RF) Module, version 4.3b, commercial software. Further, 2D PhC nanocavity was obtained by introducing point defect, where the optical field was well-confined in the defect via tuning the chemical potential of graphene in the defective region. Also, the bending wvaguide and the beam splitter are realized by introducing the line defect into 2D PhC. These results provide new insight into the designing of PhC devices, including PhC waveguides, PhC nanocavities, plasmonic filter, plasmonic switch, etc.To investigate 1D graphene-based plasmonic PhC, double-layer graphene, constituted by two graphene sheets with an interval of 0.3 nm, is periodically arranged along x direction to form the 1D PhC in our model. J = σgE along the graphene sheet [E is the electric field of the plasmon. The surface conductivity of graphene σg, constituted by the interband electron transitions σinter and the intraband electron-photon scattering σintra, is obtained from the Kubo formula [c is the chemical potential, Bk is the Boltzmann constant, T is the temperature, ω is the angular frequency of the plasmon, and τ is the electron momentum relaxation time. In this paper, we set T = 300 K, τ = 0.5 ps.The graphene sheet is treated as an ultra-thin film that can be characterized by surface conductivity in our model. Therefore, the surface current density of graphene is defined as ne sheet ,43,44, w formula ,46,47,48a = 20 nm. c1, is surrounded by the same sheet of graphene with a different chemical potential μc2. The graphene material can be characterized by effective index defined as effn = 𝛽/0k, where 0k is the wave number in free space. As for transverse magnetic (TM) mode, the propagation constants of SPPs supported by a single-layer graphene, can be expressed as [0 (377 Ω) is the intrinsic impedance of the free space. As far as band structure calculations are concerned, it is generally sufficient to consider the wave vector k around the edge of the irreducible Brillouin zone (IBZ: form Г to X to M) while ignoring the inner region, which can be justified from the symmetry of the structure.As for 2D plasmonic PhC, we use monolayer graphene with periodically modified chemical potential, to form the 2D plasmonic PhC with square lattice and lattice constant essed as ,50,51,52Similar to the electronic density of states which experience significant modifications in a semiconductor, the periodic modulation of the dielectric constant results in a photonic DOS. Photonic DOS is defined as the number of the PhC Eigen-state inside the unit frequency range. The DOS is calculated by counting all allowed states within the frequency region of an infinitesimally small interval, which is expressed as follows ,54:(5)NThe periodicity of PhCs results in PBGs where no electromagnetic modes are allowed to have frequencies in the gaps. To obtain the band structure of plasmonic PhC, we need to iterate Eigen-frequency in each k point precisely due to the frequency dependent dielectric constant which results in the analytical solutions hardly being acquired. Furthermore, we modify the width of graphene nanoribbon in the top layer to see how the band structure changes. c1 = 0.9 eV, μc2 = 0.6 eV, and the corresponding DOS. At some specific frequencies, the band gaps are overlapped forming so-called complete PBG with frequency range from 161.7 THz to 165.9 THz, which means that plasmons will not propagate inside the PhC at any angle. Moreover, the DOS within PBG is almost zero, which means there is no allowed Eigen-state in this frequency range.In the same way as for 1D plasmonic PhCs, the band structure of 2D plasmonic PhCs can be calculated for a square lattice of 2D PhC with modified chemical potential. c1 = 0.6 eV, μc2 = 0.9 eV, within all the investigated frequency ranges at least one Eigen-state exists, so no complete PBG appears. The first band covers the frequency range from 142.0 THz to 169.7 THz, the second band covers the frequency range from 164.1 THz to 184.3 THz, etc. However, considering the band structure, it is demonstrated that the PhC has wide partial PBGs at some certain propagation directions. For example, there are no Eigen-frequencies at the point M within the frequency range from 169.7 THz to 179.7 THz, which means that the light with frequency in this range propagating in the corresponding direction is forbidden.As it can be seen from c2 as 0.8 eV and 0.85 eV, respectively. Both the band position and bandwidth can be tuned distinguishedly.In order to tune the partial PBG shown in , we modic1 = 0.6 eV, μc2 = 0.9 eV, and the chemical potential of the defect is μc3. As it can be seen from As an application of graphene based plasmonic PhC proposed in this paper, the properties of the nanocavity formed by the point defect. To obtain PhC nanocavity, we introduce point defect into 3 × 3 PhCs, i.e., modifying the chemical potential of the central circle in c3 from 0.085 eV to 0.25 eV. It can be seen that the resonant frequencies are around 175 THz where the partial PBG exists (shown in −5(λ0)2 (λ0 is the free space wavelength), which is comparable to the counterpart of the graphene coated InGaAs nanowire, and much smaller than the conventional cavities [shown in , which mcavities . This na2 on Si substrate. The thickness of the SiO2 layer is periodically modified to get the chemical potential of the graphene periodical variation when external electric field is applied. So the plasmonic crystal structure is achieved. When the thickness of one site of SiO2 is different from the regular value, the different chemical potential is obtained, and finally the nano-cavity is formed.Practically, the means of fabrication of the proposed nano-cavity can follow the method proposed in Reference , where tc1 = 0.6 eV, μc2 = 0.9 eV. The path full of blue circles with chemical potential μc3 of 0.3 eV represents the line defects which we used to realize the PhC beam splitter. Another application of the 2D PhC is the beam splitter and bending waveguide. We introduce a line defect into 9 × 9 PhC. c3 of 0.3 eV represents the line defects. c1 = 0.9 eV, μc2 = 0.6 eV, and partial PBGs with frequency range from 169.7 THz to 179.7 THz for 2D PhC with μc1 = 0.6 eV, μc2 = 0.9 eV. The PhC nanocavity with resonant frequency around 175 THz was realized by introducing point defect into the 3 × 3 PhCs with an ultra-small size of 60 × 60 nm. Also, the bending wvaguide and the beam splitter are proposed by introducing the line defect in the crystal structure. The proposed graphene based PhC structure might find broad applications in the future high density plasmonic integrated circuit technique.In summary, graphene-based 1D and 2D plasmonic PhCs are proposed, where the band structure and the DOS are numerically investigated. Several PBGs of 1D PhC are found, and there is no Eigen-state within the frequency range where PBGs exist. Meanwhile, we have found complete PBGs with frequency range from 161.7 THz to 165.9 THz for 2D PhC with μ


Idiopathic Pulmonary Fibrosis (IPF) is an incurable progressive fibrotic disease of the lungs. We currently lack a systematic understanding of IPF biology and a systems approach may offer new therapeutic insights. Here, for the first time, a large volume of high throughput genomics data has been unified to derive the most common molecular signatures of IPF. A set of 39 differentially expressed genes (DEGs) was found critical to distinguish IPF. Using high confidence evidences and experimental data, system level networks for IPF were reconstructed, involving 737 DEGs found common across at least two independent studies. This all provided one of the most comprehensive molecular system views for IPF underlining the regulatory and molecular consequences associated. 56 pathways crosstalks were identified which included critical pathways with specified directionality. The associated steps gained and lost due to crosstalk during IPF were also identified. A serially connected system of five crucial genes was found, potentially controlled by nine miRNAs and eight transcription factors exclusively in IPF when compared to NSIP and Sarcoidosis. Findings from this study have been implemented into a comprehensive molecular and systems database on IPF to facilitate devising diagnostic and therapeutic solutions for this deadly disease. There is typically no response to general anti-inflammatory therapy such as glucocorticosteroids, which are effective in some IIPs such as Nonspecific Interstitial Pneumonia (NSIP). Some antifibrotic agents and adenosine receptor antagonist based solutions have shown some limited promise4. While new targets have emerged, such as proinflammatory cytokines 5, the negligible molecular systems information for IPF remains a problem. A gene expression level understanding of IPF is incomplete, with limited number of studies mostly microarray (MA) based11 and hardly a couple of RNA-seq studies13. Also, very few study has been done covering systems biological perspectives but not specific to IPF9. Limited MA based studies with non-coding RNAs have also been performed, but small RNA-seq studies are lacking16.Idiopathic interstitial pneumonias (IIPs) are interstitial lung diseases with unidentified mechanism, distinguished by matrix deposition and alveolar epithelium disruption. Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing IIP that has no effective therapy with a mortality rate higher than many cancers; median survival from the time of diagnosis being about three yearshttps://research.cchmc.org/pbge/lunggens/mainportal.html; http://montgomerylab.stanford.edu/resources.html). The present study has been carried out considering these factors. The study attempts to unify the best available gene expression data for IPF, derive its core characteristics towards generating a computational model of IPF pathology and create a comprehensive state-of-the-art database dedicated to IPF research.From a systems biology perspective, IPF offers both opportunities and challenges. The main challenge is a likely heterogeneity within IPF, which is a clinical diagnosis based on typical diffuse radiological or pathological findings that may be seen in a limited form in other IIPs or even aged lungs. Further, the IPF lung is itself heterogeneous. Thus there is likely to be substantial background noise, as evidenced by high variability between studies. This also presents an opportunity to systematically consolidate all these studies, using computational approach to extract the underlining characteristic signatures for IPF. Such efforts are critical towards better molecular characterization of IPF that could lead to better diagnosis, classification and therapy. This is important because the therapeutic response is very different between different types of IIP and possibly within different subsets of IPF. There is also a need for unified information portal and database for molecular and systems biology of IPF beyond the few existing resources on expression data reporting that have limited scope 13 have been conducted, differed in their expression measurement approaches to identify the DEGs and had very limited agreement. After implementing a common expression analysis protocol , about 70% reads could be mapped, resulting into the identification of 487 and 860 DEGs in the two studies. 124 DEGs were found common between them. However, there was no DEG common across all the gene expression studies in IPF, clearly underlining the poor consensus in this area. This was potentially related to insufficient coverage in some studies due to limited number of MA probes. For example, data from the study done by Yue et al.7 covered only 4% of the total genes in the human genome.While two prior RNA-seq studies  (referred onwards as Set A1). We further found an overlap for 176 DEGs (referred onwards as Set A2) between this RNA-seq and the study done by Cho et al.9. The intersection of Set A1 and A2 was a set of 39 DEGs (referred onwards as Set A) 13 we expected this set to be a more robust signature of IPF than the previously identified Set A113. To verify this, a comparative k-means cluster analysis was done over the sets while using Z-score transformed expression values.Because of these irregularities, the two studies performed by Yue et al.12, the clustering analysis with either set yielded only 67% accuracy. Due to very small size of this set, with three normal and three IPF individuals, this translates to only one misclassification per label. Besides this, these two different sets were also evaluated against the cases of Sarcoidosis and NSIP, the IIP diseases that are part of the differential diagnosis of IPF during clinical evaluation. Both the datasets perfectly distinguished all the cases for Sarcoidosis and NSIP from IPF  model based on the gene expression data. Details of implementations are given in the methods section and associated supplementary methods. The average accuracy over 10 different randomly built models was observed higher for Set A with 92.72% compared to 91.03% accuracy observed for Set A1 24 were considered for differential expression analysis of miRNAs in IPF, initially. However, no reliable sRNA-seq study was found for IPF. To overcome such scarcity, a strategy was employed where an RNA-seq data13 generated for longer RNA like mRNAs was utilized to indirectly derive the expression of miRNAs. Details are given in the methods section.miRNAs are one of the most important regulatory components of cell system. miRNAs like miR-29b, miR-26a and let-7 were found associated with IPF24, the finding on miRNAs is more robust and elaborated due to inclusion of data from multiple experiments including next generation sequencing. miR-539 (overexpressed) and miR-30 family (underexpressed) were obtained as targeting the maximum number of genes  in combination with TFs MYOCD and RUNX2. MMP16 is known to be involved in collagen bundle assembly which was observed as upregulated due to downregulation of miR-210 in IPF. This gives explanation for the observation from the previous study which reported that MMP16 is characteristically upregulated in IPFTF-FFLs for larger network of Set B was composed of 49 miRNA-gene, 63 TF-gene and 32 TF-miRNA interactions , and 256 nodes joined via 1,663 edges in the subnetwork of 39 genes (Set A), depicting various important nodes of multiple connectivity. Genes FZD5 along with KCNMA1 involved in repolarization of membrane potential were found as the most important nodes containing highest in-degrees in the GRN of Set A. KCNMA1 gene was observed upregulated in IPF contrary to FZD5. The observations with KCNMA1 is critical because smooth cell/myofibroblast activity is enhanced during IPF where KCNMA1 has a stake. On the other hand, FZD5 is involved in several biological pathways such as Wnt signaling, Hippo signaling and was found downregulated in IPF. Hub gene FZD5 was observed targeted through upregulated miR-155 whose upregulation was supported by a previous study in favor of Epithelial to mesenchymal transition (EMT) processCrosstalking pathways were identified by integrating gene expression and genome wide Protein Protein Interaction (PPI) data. A total of 56 pathways crosstalks were found for Set B genes  regulated by THBS138. High expression of TGFB3 induces Smad3 which in association with ATF3 represses inhibitor of differentiation (ID1)39 known to be a TGF β responsive gene which negatively regulates cell differentiation. Wnt plays an important role in IPF through binding to Frizzled receptor and further activation of β-catenin. Frizzled (FZD5) appeared downregulated in IPF due to inhibitory regulation by miR-10a*, miR-302d, miR-133b, miR-539, miR-214* and miR-34c-3p  through four potential miR-FFLs with AFF3, HOPX, ID1, KLF6 and one potential TF-FFL . Genes related to cell proliferation (CCND1 and CCND2) and matrix metallopeptidases (MMPs) involved in cell-cell adhesion appeared upregulated due to combined action of differentially downregulated miRNA (miR-210) and upregulated TFs  via two potential 3-nodes FFLs ,  and a potential 4-nodes FFL  for CCND2 and MMPs in IPF condition . Study revealed a drastic change taking place from normal to early IPF stage, clearly underlining the processes which distinguish IPF from the rest Fig. . The mathttp://14.139.59.221/ipf and http://scbb.ihbt.res.in/SCBB_dept/Software.php.The findings and information generated through this study have been converted into a state-of-the-art database and information portal, IPF Information Portal (IPFIP), the resource for this disease, freely available online at After analysis of a large volume of high throughput genomic data, a set of 39 differentially expressed genes (DEGs) emerged critical to distinguish IPF. Experimentally validated and high confidence data with multiple evidences were used to reconstruct the system model for IPF, incorporating various PPI and regulatory interactions. Careful analysis of the networks identified several critical potential FFLs as well as compromised and benefited routes in various pathways, marking the phenotype of IPF. A system of serially connected five genes, eight TFs and nine miRNAs appeared exclusive to IPF, which could be promising for therapeutic interventions. In future, the expression of associated miRNAs in this system could be controlled in order to verify their potential role as therapeutic targets for IPF to design therapeutic agents. Finally, this study has generated a state-of-the-art database on IPF where the involved components can be analyzed and visualized in a highly informative manner. This database would be very useful for any molecular systems research on IPF.40, two RNA-seq 13, three miRNA MA 24, two MAs for Sarcoidosis 42 and one MA of NSIP (GSE5774)43 (Supplementary Table 44. TOPHAT 2.1.045 was used for mapping RNA-seq reads across the human genome (hg38) with default parameters. The alignment results were saved in BAM format. CUFFLINK-CUFFDIFF tools were used for expression analysis for RNA-seq data. The work flow of IPF high-throughput data processing to generate high confidence gene sets is given in Fig. http://www.ncbi.nlm.nih.gov/geo/geo2r/) tool containing R, Biobase46, GEOquery47 and limma48 packages was used with some basic changes in Rscript. Based on the information derived from the clustering of different gene sets with varying number of DEGs (detailed in the result section), set of feature genes was formed. The relative genome wide normalized expression values of these feature genes were used for vector transformational representation of each individual sample considered in this study. Features expression transformation into Z-score for each experimental dataset was done to bring the expression values on same scale irrespective of their platform and experimental design. The classification was implemented with 5-fold cross validation using LibSVM49.Fourteen high throughput studies were included containing six IPF MAs 50 using BLASTX with the E-value threshold of 1E-05. Binding sites analysis was done using PROMO (TRANSFAC v8.3)51, CHIP-seq (ReMap)52 and manual curations. As the TFs activate the gene expression, positive expression correlation is expected between the TF-target gene pair. Z-score normalized expression values were used to find the correlation between TF and target genes. For selecting the significantly enriched categories, p-value cut off (0.05) with Benjamini-Hochberg test adjustment was applied.DEGs having TF activity were identified. Sequences of DEGs were downloaded from Ensembl and were searched for homology in UniProt and AnimalTFDB 2.02fold change (> = 1) considering Reads Per Kilobase of transcript per Million (RPKM) values in both conditions, whereas differentially expressed miRNAs (DEmiRs) from MA studies were selected using GEO2R . miRNAs which were having at-least two or more studies with same differential expression status were taken as final set of DEmiR. miRNAs from RNA-seq approach were preferred over MA in case of contradictory differential expression status  for each miRNA-target. A work flow for miRNA analysis is provided in Supplementary Figure miRNA expression analysis was done using four high throughput studies, mentioned above. Interestingly, so far, no small RNA-seq study has been done for miRNAs with respect to IPF. In this study the RNA-seq reads were utilized to detect the active miRNA regions of genome. The overlapped reads with miRNA precursor regions (taken from miRBase V21) were expected to express along with other coding genes. Such mapping reads were capable to provide the expression information of the precursor miRNAs, which in turn indicate the expression level of mature miRNAs. Differentially expressed miRNAs were selected using logk-threshold of 3 using clique percolation algorithm, CFinder55. Potential FFLs are made of interconnected complete subgraph of TFs, differentially expressed miRNAs and genes. Variations in the type of potential FFL occur due to regulatory interaction between TFs and miRNAs. The work flow for the construction of TF-miRNA mediated regulatory network and statistics of three and four node FFLs for the three sets is illustrated in Supplementary Figure Regulatory relationships in form of miRNA-gene, miRNA-TF, TF-gene and TF-miRNA were considered for generation of regulatory network to unveil disease mechanism in IPF. Differentially expressed miRNAs, genes in IPF condition with respect to normal patient and TFs controlling both were retrieved for deciphering all such relationships. All types of interaction pairs were merged and unified to construct IPF-specific regulatory network. TF and miRNA regulatory network modules having various potential FFLs were derived at 56 R-package. The degree was kept same as for real regulatory network. All potential FFLs in each of the regulatory network were statistically analyzed for their significance while comparing to 1000 randomized regulatory networks. Only those FFLs were considered further for the depiction of regulatory mechanism in IPF which were found statistically significant at 5% level.IPF specific potential FFLs were evaluated for their significance in comparison to randomized networks. For this purpose, we compared real regulatory networks to randomized ones, preserving same degree as for the real one to estimate the probability that an FFL appears in the randomized networks. 1000 randomized networks were built, implementing edge swapping algorithm present in igraph57 was used for network visualization and analysis of various network parameters such as degree connectivity, betweenness and closeness centrality etc. Hub miRNAs and TFs were identified by sorting their outdegrees in descending order and top five hub components in each category were separated. Hub IPF genes were determined on the basis of indegree distribution.Network properties of built TF-miRNA derived regulatory networks were extracted as a consequence of their significance. Cytoscape version 3.158 of functional protein interaction. A global pathway crosstalk approach was utilized to decipher IPF mediated pathway crosstalk59. To determine the background distribution, each pathway was randomized via shuffling of genes with genes having same degree in PPI database. Randomization process was performed 1000 times and protein interaction count was calculated for all randomized pathway pairs. Protein pairs which scored significant p-value against the random model pair occurrence were considered as cross talk pairs.Set B DEGs were utilized for pathway crosstalk network reconstruction. Pathways information related to such genes were retrieved from KEGG database. Human protein interaction data was downloaded from STRING v10 repository60. Gene sets were extracted for benefited and compromised system chains due to pathway crosstalk in the disease condition. Detailed illustration representing pathway crosstalk network analysis pipeline in IPF is shown in Fig. The obtained significant pathway pairs in crosstalk were filtered for those pairs having differentially up regulated genes in both pathways. First neighbors of these DEGs in STRING database containing confidence of 500  were chosen for further analysis. Gene ontology enrichment analysis was done using GO-TermFinderThe portal is implemented using advance libraries of HTML5. Portal homepage and background pages are developed in HTML5/CSS, JQuery/JavaScript with support of Bootstrap packages. Portal work flow is described in Supplementary Figure Supplementary InformationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3


Our proof-of-concept results pave the way towards on-chip realization of efficient graphene-based active plasmonic waveguide devices for optical communications.The unique optical and electronic properties of graphene make possible the fabrication of novel optoelectronic devices. One of the most exciting graphene characteristics is the tunability by gating which allows one to realize active optical devices. While several types of graphene-based photonic modulators have already been demonstrated, the potential of combining the versatility of graphene with subwavelength field confinement of plasmonic waveguides remains largely unexplored. Here we report fabrication and study of hybrid graphene–plasmonic waveguide modulators. We consider several types of modulators and identify the most promising one for telecom applications. The modulator working at the telecom range is demonstrated, showing a modulation depth of >0.03 dB μm The combination of graphene with plasmonic waveguides remains largely unexplored. Here, Ansell et al. report the fabrication of hybrid graphene plasmonic waveguide modulators working in the telecom range, with a modulation depth greater than 0.03 dB μm−1 and with comparable characteristics to silicon-based devices. For waveguide modulators, in which a waveguide mode is propagating along and confined near a graphene plane (and which are the most promising for on-chip information processing), the portion of the waveguide mode interacting with the graphene layer is very limitedde field . For typn) and local field enhancement , which would result in higher modulation depth. There are two main problems in combining graphene and plasmonic waveguides to achieve optical modulation by gating: (i) the presence of metal layers complicates the task of gating with the spacer dielectrics being often affected by both high electric fields−1 for strongly confined SPP modes , see All three studied plasmon–polariton modes—flat plasmons (FPs), corrugated plasmons (CPs) and wedge plasmons (WPs)—can be excited by moving the incident light beam to different parts of the coupler . An opti32λ=1.5 μm. We measured the dynamic response of our modulators by applying an offset square-wave voltage to the back gate with peak-to-peak amplitude VCNP≈2.7 V; for the CP mode this is VCNP≈0.9 V). For other samples the modulation for FP modes compared with CP modes was even less pronounced  to the metal nanowire covered with the flat graphene layer (∼0.3 dB μm−1), that is, when exploiting the effect of metal surface curvature resulting in non-zero projections of strong transverse SPP fields on the graphene plane. The largest modulation depth of 0.03 dB μm−1 achieved in our hybrid graphene–plasmonic waveguides is lower than that (0.1 dB μm−1) reported for the hybrid graphene–silicon waveguidesThe WP mode provided us with the greatest modulation depths, achieved when probing the top edge of the waveguide, see inset α is the fine structure constant, Leff is an effective size of plasmon mode, σ is the gate-dependent graphene optical conductivity and σ0 is the universal graphene optical conductivityP=P0exp(−αgrl), where l is the length of the waveguide. Using the Kubo expression for graphene conductivity and writing Fermi energy as Vg=Vg−VCNP, vF is the Fermi speed in graphene and C is the gate capacitance, we were able to model the modulation data obtained in our experiments. The modelling results . Note that the achieved modulation depth exceeds by ∼50% the intensity modulation depth corresponding to the highest phase modulation demonstrated with the state-of-the-art plasmonic modulator1In conclusion, exploiting the enhancement of in-graphene-plane plasmonic fields provided by the wedge SPP waveguide configuration, we have demonstrated a prospective hybrid graphene–plasmonic waveguide modulator working at telecom wavelengths with reasonably high modulation depth of >0.03 dB μmThe use of wedge SPP waveguide configuration for light modulation by graphene gating thereby paves the way towards practical realization of very compact and efficient, potentially ultrafastThe gold plasmonic waveguides were fabricated in two lithography steps. In the first step of electron-beam lithography (and subsequent lift-off), we fabricated a plasmonic waveguide—a golden strip of 110 nm thickness, 100 μm length and 60 μm width—and the de-coupler, which was formed of a transparent gold grating for the period of 1.24 μm on the top of a quartz substrate. In the second step, we fabricated periodic lateral ridges along the bottom half of the waveguide  and the coupling non-transparent gold grating. The coupling grating was optimized for 1.5 μm wavelength normal incident light. This configuration of non-transparent coupling grating and transparent de-coupling grating allows one to excite the plasmon from one side and to measure the transmitted light from the other side, decreasing the crosstalk between input and output lights. Plasmons propagating along the nanostructured half of the waveguide, referred to as CP, hop over the ridges (arranged in a 600-nm-period grating) and generate in-plane electric fields, whereas along the planar section the evanescent electric field associated with the propagating FPs remains mostly transverse . We alsot≈50 and 70 nm and nanostructure ridge widths of w=300 and 200 nm, respectively. Both had a nanostructure ridge pitch of 600 nm.To fabricate the modulation unit on the top of plasmonic waveguides, we applied the micromechanical cleavage technique to produce graphene and hBN flakes on oxidized silicon wafers. Then, hBN crystals and graphene were wet-transferred and aligned on to the waveguide. Oxygen plasma was used to shape graphene to achieve regular geometry of flakes across the waveguide. Here hBN acts as an atomically flat substrate for graphene as well as the gate dielectric. The gold waveguide was used both as the carrier of propagating plasmons and backgate for graphene gating. Finally, we fabricated electrical contacts to graphene  and to the waveguide/backgate using photolithography. In this study we provide results obtained on two particular devices, with hBN thicknesses How to cite this article: Ansell, D. et al. Hybrid graphene plasmonic waveguide modulators. Nat. Commun. 6:8846 doi: 10.1038/ncomms9846 (2015).Supplementary Figures 1-5, Supplementary Discussion and Supplementary References.


Chemical reduction of graphene oxide (GO) to graphene employs the use of toxic and environmentally harmful reducing agents, hindering mass production of graphene which is of tremendous technological importance. In this study we report a green approach to the synthesis of graphene, bio-reduced by crude polysaccharide. The polysaccharide reduces exfoliated GO to graphene at room temperature in an aqueous medium. Transmission electron microscopy image provides clear evidence for the formation of few layer graphene. Characterization of the resulting polysaccharide reduced GO by Raman spectroscopy, Fourier transform infrared spectroscopy and Energy dispersive X-ray analysis confirms reduction of GO to graphene. We also investigated the degree of biosafety of the reduced GO and found it to be safe under 100 μg/ml. Due to the electronic, mechanical, thermal and optical uniqueness, two-dimensional graphene nanosheets (GNS) have significantly transformed areas of nanoscience ,2. Even Carbon based nanoparticles, mainly graphite nanoformulations have seized a lot of fancy recently. Ground breaking progress is being made with these formulations and the potentials are infinite. Lately, some researchers have been able to synthesize an ultra light weight Carbon Microtube, Aerographite , with exin vitro studies on BEAS-2B cells . This measures the extent of DNA damage induced by the chemical being tested. Images of 150 (50 cells/replica × 3 replicates) cells per concentration were analyzed for human lymphocytes. The median values of each concentration with respect to the comet parameter were calculated.6 cells) of PBS containing 25 μM DCFHDA. A set of three replicates per concentration were maintained for the experiment. After 15 minutes incubation at room temperature in the dark, the DCF fluorescence in cells was measured by flow cytometry  and 20,000 events were acquired for analysis. Results were expressed as the mean fluorescence intensity. The interference of nanoparticles in the assays was eliminated by maintaining sample blanks for all the concentrations tested. H2O2  treated cells were used as positive control for the experiment.Following treatment with PR-GO nanoparticles  for 3 h at 37°C, cells were washed twice with PBS and re-suspended in 500 μl . Fresh blood samples for lymphocyte culture were obtained from healthy volunteers after obtaining written informed consent. Subsequent experiments were performed in vitro and did not involve any form of human intervention or studies in animal model and hence no additional clearance was required. The Bioethics Committee for Animal and Human Research Studies, University of Calcutta was fully aware of and sanctioned this present research work.P. flabellatus, gave a yield of 4.5 ± 0.51% dry weight. it consisted mainly of carbohydrate (43.75 ± 3.25%) with low amount of protein (12.5%). No phenols were detected in the polysaccharide. Total glucan content of the polysaccharide was 17.68 ± 1.89% among which 1.29 ± 0.31% were α-glucans and 16.39 ± 2.2% were β-glucans. To further investigate the physical structure of polysaccharide, Congo red assay was performed which showed a bathocromic shift from 491 nm to 500 nm , polysaccharide solution (PS) and the reaction mixture of the polysaccharide with graphene oxide respectively. Only the reaction mixture displayed a time dependent colour-change, while the polysaccharide and the graphene oxide solutions retained their original colours . the iniParticle size of PR-GO was determined by dynamic light scattering measurement. The size of the particles were found to be in the range of 15 to 155 nm . The sizTEM image shown in X-ray diffraction study was used to confirm the reduction of graphene oxide to crystalline graphene. XRD pattern that was obtained has been represented in the The Energy-dispersive X-ray (EDX) spectrum  recordedPleurotus flabellatus have been shown in -1 due to the presence of O-H stretching [-1 were owing to the absorption of the polysaccharide extract containing phenolic compounds on the surface of graphene. So, from the above observation it is clear that the increased reduction time leads to a greater removal of the oxygen functionalities from the GO surface, indicating a successful reduction of GO to PRGO [-1 attributed to the aromatic C = C groups present in all three spectra. This suggests that the frame of sp2 carbon atoms after reduction by crude polysaccharides was retained well, as before [-1 in case of GO, while they were completely absent in the spectra of PR-GO as well as crude polysaccharides [-1 and 1060 cm-1, found in the spectra of GO and PR-GO nanoparticles, signified the epoxy C-O and alkoxy C-O stretching vibration, respectively [-1 was due to N-H deformation [-1 (PR-GO spectra) due to the symmetric stretching of the carboxyl side groups of the polysaccharide molecules [-1 of the polysaccharide spectrum indicated C-C and C-O stretching vibrations in pyranoid rings indicating pyranose form of sugar [-1 signified the presence of R–CH groups [-1 signifies the presence of β-glycosidic bond and therefore indicated the existence of β-glucan. These analyses further corroborated our inference that the polysaccharide consists mainly of β-structures of pyranose sugars and that the interaction of GO with the crude polysaccharide formed PR-GO nanosheets.Fourier transform infrared spectroscopy deals with the stretching, bending, bonding and wiggle responses of molecular species in a given sample specimen. FTIR absorption spectra of the biosynthesized, vacuum dried GO, PR-GO nanosheets and crude polysaccharide of retching . The pre to PRGO . Howevers before . The chacharides . The FTIectively . Howeverectively ,60. Bandormation  found inolecules . This inof sugar ,68. The H groups . In the -1 in the Raman scattered light observed for both GO and PR-GO are crucial for this purpose  at treatment dose 250 μg/ml. Treatment doses 50 and 100 μg/ml induced DNA damage, but was not statistically significant when compared to control (2O2 (% Tail DNA: 55±8) and was used as a positive control for the experiment.In addition to cytotoxicity, genotoxicity of the particles was evaluated using comet assay. Genotoxicity assessment of particle could provide valuable information regarding DNA damage that might lead to better understanding of mutation and cancer. Genotoxicity of various graphene oxides has been reported by some authors –78. In t control . Signifi˷1.8-fold increase in ROS generation at the highest concentration tested (250 μg/ml), and ˷6-fold increase in the positive control (H2O2). In a study by Chang et al. (2011), the authors suggested of a dose-dependent oxidative stress in A549 cells, despite little/absence of cytotoxic response. In another study [In addition to direct DNA damage, certain nanoparticles are known to induce oxidative stress. Flow cytometric analysis indicated concentration dependent increase in ROS production . There wer study  the authIn conclusion, a green approach to the synthesis of graphene nanosheets is reported using exfoliated graphene oxide as the precursor and a crude fungal polysaccharide as the reducer. The use of an environment-friendly reducing and capping agent is what this method derives its merit from. The method should find practical applications in bulk-synthesis of graphene nanosheets. Also, from all the toxicity endpoints studied in human PBMCs, this newly synthesized nanoparticle could be considered biologically safe at concentrations below 100 μg/ml.


The properties of graphene are strongly affected by metal adsorbates and clusters on graphene. Here, we study the effect of a thin layer of platinum (Pt) metal on exfoliated single, bi- and trilayer graphene and on chemical vapor deposition-grown single-layer graphene by using Raman spectroscopy and transport measurements. The Raman spectra and transport measurements show that Pt affects the structure as well as the electronic properties of graphene. The shift of peak frequencies, intensities and widths of the Raman bands were analyzed after the deposition of Pt with different thicknesses  on the graphene. The shifts in the G and 2D peak positions of the Raman spectra indicate the n-type doping effect by the Pt metal. The doping effect was also confirmed by gate-voltage dependent resistivity measurements. The doping effect by the Pt metal is stable under ambient conditions, and the doping intensity increases with the increasing Pt deposition without inducing a severe degradation of the charge carrier mobility. HowevIn this paper, we report the effect of a thin layer of Pt on exfoliated single-, bi- and trilayer graphene as well as on single-layer graphene grown by chemical vapor deposition (CVD) using Raman spectroscopy and transport measurements. The Raman spectra and transport measurements show that Pt affects the structure as well as the electronic properties of graphene. The Raman bands were analyzed before/after the deposition of Pt with different thicknesses  on graphene. The Raman spectra and transport measurements indicate the n-type doping effect by Pt deposition.2.−7 torr) at a rate of 1.0 Å s−1 The Pt deposition was monitored by a quartz oscillator and confirmed with atomic force microscopy (AFM). To avoid sample heating during deposition, the sample stage was cooled by a continuous water flow. The Raman spectra were measured before and after the metal deposition on the same graphene sample. The Raman spectra were recorded at room temperature with a Renishaw microspectrometer. The laser wavelength was 514 nm; to avoid a laser-induced heating effect, the power was kept at 1.0 mW. The diameter of the laser spot was less than 1 μm. The Dirac points  of the graphene samples were observed by a gate-voltage dependent resistivity measurement using a 4-probe configuration method with a lock-in amplifier at room temperature.Pt metals with different thicknesses  were deposited on exfoliated single, bi- and trilayer graphene and on single-layer CVD-grown graphene by a thermal evaporation system in a high vacuum (∼8 × 10μm thick copper foils from Alfa Aesar (99.8% pure) via thermal CVD. A mechanically polished and electropolished copper foil was inserted into a thermal CVD furnace. The furnace was evacuated to ∼10–4 torr and heated to 1010 °C under an H2 gas flow (∼10–2 torr). After the temperature became stable at 1010 °C, both the CH4 (20 standard cubic centimeters per minute (sccm)) and the H2 (5 sccm) were injected into the furnace for 8 min to synthesize the graphene. After the graphene synthesis, the sample was cooled at a rate of 50 °C min−1 to room temperature [2/Si wafer as follows: The Cu foil was etched in an aqueous solution of ammonium persulfate (APS). The surface of the graphene was spin-coated with polymethyl methacrylate (PMMA), and the sample was then baked at 70 °C for 10 min The PMMA coating was applied to prevent the graphene films from cracking and folding during the transfer to a desired substrate. The PMMA/graphene film was washed with deionized water after the Cu foil had been completely dissolved; it was then transferred onto the Si/SiO2 wafer. The PMMA film was removed with acetone. The graphene sample was subsequently cleaned in isopropanol and dried in a N2 flow [The exfoliated single, bi- and trilayer graphene were obtained from the bulk graphite using a standard Scotch Tape method and were confirmed with optical and Raman spectra, whereas the CVD-grown single-layer graphene was obtained using thermal CVD. The growth process of the graphene is as follows: The graphene used in this study was grown on 25 perature , 16. The N2 flow , 16.3.μm. The line profiles of the graphene sample after 1, 3 and 5 nm Pt deposition are shown in figure Figure The Pt film’s morphology is examined in figures −1 and 2683 cm−1, respectively, as shown in figures ID/IG ratio increases in the exfoliated SLG from 0.01 to 0.3 after the deposition of 1 nm of Pt. The ratio of ID2/IG becomes 2.39 compared to 3.30 in pristine SLG. After the 3 nm Pt deposition on the exfoliated SLG, the ratio of the ID2/IG is significantly reduced to 1.86. The ratio of the ID/IG is 0.41 for the exfoliated SLG after 3 nm of Pt metal deposition. The 2D band is a single peak at 2671 cm−1, which is red- (downward) shifted by 13 cm−1 from the pristine graphene. On the other hand, the G band of the graphene after the Pt deposition is found to be at 1587 cm−1, which is blue- (upward) shifted by 6 cm−1. After the 5 nm Pt metal deposition, a more pronounced D band peak is observed. The ratio of ID/IG is 0.50 for the graphene after 5 nm of Pt deposition. The intensity of the 2D band is significantly reduced, and the ratio of the ID2/IG becomes 1.25. The 2D band is observed at 2665 cm−1, which is red-shifted by 19 cm−1 from the pristine graphene. The FWHM of the G peak has been observed at 15.5, 13.1, 10.2 and 9.7 cm−1 before and after the 1, 3 and 5 nm Pt metal deposition, respectively. The blue shift of the G peak, the red shift in the 2D peak and the reduction of the FWHM of the G peak indicate an n-doping effect [−1 for the pristine CVD-grown SLG, whereas it was found at 1585, 1588 and 1591 cm−1 for the 1, 3 and 5 nm Pt deposition, respectively. The ratio of ID/IG was changed to 0.28, 0.37 and 0.47 after the 1, 3 and 5 nm Pt deposition, respectively, as compared to the 0.02 ratio for the pristine CVD-grown graphene. The 2D band of the pristine graphene was observed at 2684 cm−1, whereas those after the Pt metals with thicknesses of 1, 3 and 5 nm were observed at 2679, 2674 and 2668 cm−1, respectively, which is red-shifted by 16 cm−1 from the pristine CVD-grown graphene. The ratio of ID2/IG was changed to 2.3, 1.9 and 1.35 after the 1, 3 and 5 nm Pt deposition, respectively, compared to the 2.9 ratio for the pristine CVD-grown graphene.The Raman spectra of the exfoliated and CVD-grown SLG before and after the 1, 3 and 5 nm Pt deposition are shown in figure g effect –21, whic−1 before and after the 1, 3 and 5 nm Pt metal deposition, respectively. We found that the FWHM of the G-peak decreased after the Pt deposition. However, the strain usually results in an increase of the FWHM of the G-peak, as reported in [Both the n- and p-type doping can lead to the reduction of the FWHM of the G-peak, as already reported in . We haveorted in . Furtherorted in . Howeverorted in –21. We n−1 for the exfoliated BLG. On the other hand, the G band of the bilayer graphene after the 1, 3 and 5 nm Pt deposition was found to be at 1583 cm−1, 1586 cm−1 and 1589 cm−1. The ratio of I2D/IG became 0.90 after the 5 nm Pt deposition, whereas it was 1.36 in the pristine BLG. The 2D peak was found at 2705 cm−1, 2701 cm−1 and 2696 cm−1 after the 1, 3 and 5 nm Pt deposition, respectively. The red shift of 11 cm−1 in the 2D band was found in the exfoliated BLG after the 5 nm Pt deposition. It is known that the blue shift of the G band positions and the red shift of the 2D band positions indicate the n-doping of the graphene. Figures −1 for the exfoliated TLG. On the other hand, the G band of the graphene after the 1, 3 and 5 nm Pt deposition was at 1584, 1585.5 and 1587 cm−1. The ratio of ID2/IG became 0.65 compared to 0.8 in pristine TLG. The red-shifted 2D peaks were found at 2706 cm−1, 2703 cm−1 and 2699 cm−1 after the 1, 3 and 5 nm Pt deposition. Again, the blue shift of the G band positions and the red shift of the 2D band positions are indicative of n-doping of the TLG [Figures  the TLG –21. The ID/IG ratio before and after the 1, 3 and 5 nm Pt deposition in exfoliated SLG, BLG and TLG. The ID/IG ratio for the pristine state in SLG, BLG and TLG is around zero, indicating the high quality of the graphene samples, but after the Pt deposition, the ID/IG ratio increases significantly. A larger change is observed for the SLG compared to the BLG and TLG. The ID/IG ratio is 0.5 in the SLG, while it is 0.25 in the TLG. Figure ID2/IG ratio before and after the 1, 3 and 5 nm Pt metal deposition in exfoliated SLG, BLG and TLG. The largest change in the ID2/IG ratio is observed in the SLG. The ID2/IG ratio changes from 3.1 to 1.1 in the SLG, while the ID2/IG ratio changes from 0.8 to 0.65 in the TLG.Figure g), which corresponds to the Dirac point, while the slope indicates the mobility of the charge carriers in the graphene [Vg = 0 V, indicating an undoped feature of the pristine graphene. The deposition of Pt on the graphene surface causes the shift of the Dirac point toward negative gate-voltages, showing the n-type doping effect [Figure graphene –25. The g effect , 27. Theμ = (1/Cg)(∂σ/∂Vg), where σ is the conductivity of the samples, and Vg is the gate-voltage [Figure -voltage –31. The 4.Using Raman spectroscopy and transport measurements, we studied the interaction of thin layers of Pt with exfoliated single, bi- and trilayer graphene and CVD-grown single-layer graphene. The Raman spectra and transport measurements indicate that Pt affects the structural as well as the electronic properties of the graphene. The shifts in the G and 2D peak positions indicate the n-doping of graphene by the Pt metal. The doping effect is also confirmed by gate-voltage dependent resistivity measurements. However, it was found that Pt affects the characteristics of the SLG more than the BLG or TLG. We have also verified the stability of our graphene devices under ambient conditions and in an oxygen flow.


A pilot clinical study was performed on seven dogs with oral malignant melanoma (OMM) and two with undifferentiated sarcoma. Objective antitumour responses were observed in one dog with OMM  and one with undifferentiated sarcoma  when c4G12 was given at 2 or 5 mg/kg, every 2 weeks. c4G12 could be a safe and effective treatment option for canine cancers.Immunotherapy targeting immune checkpoint molecules, programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1), using therapeutic antibodies has been widely used for some human malignancies in the last 5 years. A costimulatory receptor, PD-1, is expressed on T cells and suppresses effector functions when it binds to its ligand, PD-L1. Aberrant PD-L1 expression is reported in various human cancers and is considered an immune escape mechanism. Antibodies blocking the PD-1/PD-L1 axis induce antitumour responses in patients with malignant melanoma and other cancers. In dogs, no such clinical studies have been performed to date because of the lack of therapeutic antibodies that can be used in dogs. In this study, the immunomodulatory effects of c4G12, a canine-chimerised anti-PD-L1 monoclonal antibody, were evaluated  Current treatment for dog cancers includes surgery, chemotherapy and radiotherapy. In addition to these, some molecular-targeted therapies including tyrosine kinase inhibitors are available for treatment of a limited number of cancer types such as mast cell tumour2. Many attempts have been made to find new options for dog cancer treatment and some targeted antibodies have been developed and tested in vitro and in vivo4.Cancer is a common cause of death in dogs6. Of particular note, anti-PD-1 antibody has shown promising anticancer activity with tolerable toxicity profiles in a number of clinical trials7, altering the standard of treatment for melanoma, non-small cell lung cancer and other cancers. However, there is no such immunotherapy available to date for treatment of dog cancers, limiting treatment options particularly when systemic therapy is needed to control metastatic disease.In human cancer treatment, immunotherapy has gained attention partially due to the success in some agents targeting immune checkpoint molecules such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4)8, and aberrant PD-L1 expression in tumour cells or other cells in the tumour microenvironment has been demonstrated in various cancer types10. Blocking the PD-1/PD-L1 pathway with anti-PD-1 or PD-L1 antibody restores multiple effector functions of antigen–specific T cells11, with subsequent remission of cancer12. This approach is considered a promising immunotherapy that targets the immune evasion mechanisms of cancer cells. In dogs, PD-L1 expression was observed in various cancer types including oral malignant melanoma (OMM), hemangiosarcoma and osteosarcoma. PD-1 was significantly upregulated in tumour-infiltrating lymphocytes obtained from OMM13. However, the therapeutic potential of the PD-1/PD-L1 blockade has not yet been fully elucidated because of the lack of therapeutic antibody against these molecules that can be used in clinical experiments with dogs.PD-1 is a costimulatory receptor expressed mainly on activated T cells. It suppresses T-cell activation upon binding to its ligands, PD-ligand 1 (PD-L1) and PD-L2. In humans, the expression of PD-1 is upregulated in tumour antigen–specific T cells15. For clinical purposes, rat mAb needs to be canine-chimerised to reduce immunogenicity and to make it function properly in a dog’s body. By converting the constant regions of the rat mAb to those of canine antibodies, a large part of the mAb is then of canine origin, while the variable regions remain intact, thus conserving the binding properties of the antibody. This chimeric mAb acquires effector functions which depend on the Fc region of the mAb, including antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. However, because those two functions are not required for a therapeutic anti-PD-L1 antibody, canine IgGD  can be used as a heavy chain constant region to minimise possible side effects caused by the cytotoxicity of mAbs17.Recently, rat monoclonal antibodies (mAbs) which recognise dog PD-L1 have been established in our laboratoryin vitro using recombinant protein–based assays. Furthermore, the clinical efficacy of the chimeric mAb was evaluated in dogs with OMM or undifferentiated sarcoma, which might express PD-L1 as a possible mechanism of immune escape15. Here we provide the first evidence of clinical benefit of anti-PD-L1 mAb in dogs, suggesting that it could be a novel option for canine cancer treatment.In this study, rat-dog chimeric anti-PD-L1 mAb was prepared and tested for its binding and blocking property 14. In the blocking assay of canine PD-1/PD-L1 binding, 6G7 (rat IgM) and 4G12 (rat IgG2a) showed efficient blocking ability, while 5A2 (rat IgG1) did not have an apparent blocking effect -DG44 cells were transfected with the c4G12 expression vector and high-producer cell clones were established by the dihydrofolate reductase (DHFR)/methotrexate (MTX) method. The expression and purification of c4G12 was confirmed by SDS-PAGE analysis Fig. . The purD-1 Fig.  and cCD8D-1 Fig. , and posTo assess the immunomodulatory function of c4G12, dog peripheral blood mononuclear cells (PBMCs) were stimulated with the superantigen, staphylococcal enterotoxin B, in the presence or absence of c4G12. Cytokine production and cell proliferation were evaluated on days 3 and 2, respectively. Treatment with c4G12 significantly enhanced interleukin (IL)-2 and interferon (IFN)-γ production from dog PBMCs while cPD-L1-Ig treatment suppressed the production of IL-2 Fig. . The proetc.) were noted during the study, they were not considered treatment-related, and additional medication or cancellation of c4G12 treatment was not indicated.Seven dogs histologically diagnosed with OMM and two dogs with undifferentiated sarcoma were enrolled in the pilot clinical trial, after the confirmation of PD-L1 expression in the primary cancers by immunohistochemistry . The treatment group comprised two miniature dachshunds, a pug and a golden retriever, with a median age at the time of confirmation of pulmonary metastasis of 12.5 years . The survival pattern of treatment group and that of control group was analysed by Kaplan-Meier method and the estimated median survival of treatment group and control group were 93.5 days  and 54 days , respectively .Among dogs with OMM treated with c4G12, four dogs with pulmonary metastasis had progression of the disease (PD). However, one dog had unexpectedly long survival  after the confirmation of pulmonary metastasis. Because reliable survival data for dogs with stage IV OMM (with confirmed distant metastasis) has not been reported in the literature to date, survival from the confirmation of pulmonary metastasis to death was retrospectively investigated in dogs with OMM treated at our veterinary hospital from 2013 to 2016, and the survival pattern of treatment group was compared to that of historical control group. The control group comprised nine miniature dachshunds, two Labrador retrievers, a Chihuahua, a golden retriever, a beagle, a Pomeranian, a miniature schnauzer and a mixed breed. The median age at the time of confirmation of pulmonary metastasis was 14 years . Development of alternative or additional systemic therapy is needed for the control of OMM, especially when it has metastasised.Malignant melanoma is a cancer derived from melanocytes, which may occur in the oral cavity, lip, skin, digit or eye in dogs23. However, in the field of veterinary science, little is known about the association between these molecules and animal diseases. We previously reported that PD-1 and PD-L1 were upregulated in chronic bovine infections such as bovine leukemia virus infection, Johne’s disease and bovine anaplasmosis, and the blockade of those molecules by specific mAbs associated with enhanced immune response27. In dogs, our group and others have reported PD-1 and PD-L1 expression in various cancer types, and the blockade by several mAbs restored the immune cell function in vitro31. However, the therapeutic potential of those mAbs has not been reported in vivo because rat or mouse mAb may not be suitable for repeated administration to dogs for several reasons. First, because rodent protein itself is immunogenic for dogs, severe allergic reactions including anaphylaxis may occur. Second, an antibody response can be established against the administrated mAb itself, which may neutralise and reduce its therapeutic activity. To overcome these shortcomings, mAbs have been chimerised to prepare therapeutic antibodies for humans. For example, rituximab, anti-CD20 mAb for the treatment of non-Hodgkin’s lymphoma, is a mouse-human chimeric mAb and is widely used with tolerable side effect profiles32. Although humanised or fully humanised mAb has been developed and is considered a better option for therapeutic mAbs for humans, chimeric mAb is still a fascinating option for veterinary species because it does not need conformational analysis of animal antibodies or specialised mice genetically modified to produce animal antibodies. In fact, canine chimerisation of rat mAb was done quite simply in this study by combining the nucleotide sequence of the rat mAb variable regions with the dog mAb constant regions. Chimerisation did not affect the binding and blocking property of mAb when rat IgG was converted into dog IgG, and the chimeric mAb was well tolerated in dogs in the pilot clinical study, with no evidence of allergic reactions. Therefore, we concluded that chimerization of antibody is a simple and effective strategy to prepare therapeutic mAbs for dogs, although it should be validated in future studies in which a larger number of dogs or different chimeric mAbs are included in the evaluation.Immunotherapy targeting immune checkpoint molecules such as PD-1 and PD-L1 has shown great promise for various cancer treatment in human and mouse studies22, our result seems consistent with human studies. In addition, the c4G12 treatment may have prolonged survival in dogs with OMM with pulmonary metastasis, although statistical significance was not reached (p = 0.10), possibly because the sample size was too small (treatment group n = 4). Considering that dogs with OMM with pulmonary metastasis have quite a poor prognosis with standard therapies, c4G12 could be a novel treatment option for at least palliative purposes. Because the proof of concept has been established in this pilot clinical study, further clinical studies should be performed to fully elucidate the clinical benefit of c4G12.To the best of our knowledge, this is the first report in which canine chimeric anti-PD-L1 mAb was prepared and tested for its clinical efficacy in dogs with malignant cancers. Treatment with c4G12 induced an obvious antitumour response in a dog with OMM and in another with undifferentiated sarcoma. The objective response rate in OMM was 14.3% (1/7) and that in undifferentiated sarcoma dogs was 50.0% (1/2). Because objective response rate of anti-PD-L1 mAb in melanoma patients was 17.3% (9/52) in a human clinical trial5. While both cell surface and intracellular expression of PD-L1 was observed in the tumour cells . In humans, side effects possibly caused by the PD-1/PD-L1 inhibiting–mAbs include infusion-related reactions, hypersensitivity, pneumonitis, colitis, diarrhoea and hypothyroidism36. Therefore, further studies may be beneficial not only for developing dog cancer treatment but also to inform human preclinical studies using PD-1/PD-L1 inhibiting–mAbs.In conclusion, we have successfully developed a canine chimeric anti-PD-L1 mAb and tested its clinical efficacy in dogs with OMM or undifferentiated sarcoma. The pilot clinical study demonstrated the safety and antitumour activity of anti-PD-L1 mAb in dogs. Some dog cancers, including malignant melanoma, are considered preferable preclinical models of human cancer research with similar biological behaviours, resistance to chemo/radiotherapy, metastatic propensity and spontaneous occurrence in outbred and immunocompetent settingsAnimal use throughout this study was approved by the Institutional Animal Care and Use Committee, Hokkaido University  and the Graduate School of Veterinary Medicine, Hokkaido University . All experiments were performed in accordance with relevant guidelines and regulations of the Graduate School of Veterinary Medicine, Hokkaido University, which has been fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Peripheral blood samples of clinically healthy dogs were collected from 1- to 3-year-old beagles kept at the Experimental Animal Facility, Graduate School of Veterinary Medicine, Hokkaido University. Dogs with OMM or undifferentiated sarcoma seen at the Veterinary Teaching Hospital of Hokkaido University were enrolled in a pilot clinical study of the chimeric antibody. Written informed consent was obtained from the dogs’ owners and veterinarians.2, 125 rpm) in CD OptiCHO medium or in Dynamis medium (Life Technologies) supplemented with 2 mM GlutaMAX I. For the culture in Dynamis medium, 4 g/L, 4 g/L and 6 g/L glucose was added on days 3, 5 and 7, respectively, and 3.3% v/v EfficientFeed B + (Life Technologies) reconstituted at 3 × concentration was added on days 3, 5, 7 and 10. Purification of chimeric mAb from the culture supernatant was performed by affinity chromatography using Ab-Capcher Extra , and the buffer was exchanged with phosphate-buffered saline (PBS) by ultrafiltration using Amicon Ultra-15 Ultracel-50 . The concentration of c4G12 was measured by Nanodrop 8000 Spectrophotometer . To confirm the expression and purification of c4G12, SDS-PAGE was performed in reducing or non-reducing conditions using SuperSep Ace 5–20% gradient gel  and 2× Laemmli Sample Buffer . Precision Plus Protein All Blue Standards (Bio-Rad) was used as a molecular-weight size marker, and proteins were visualised with Quick-CBB (Wako). The purity of c4G12 was evaluated by densitometry using CS Analyzer version 3.0 software  and was routinely >90%. For SDS-PAGE analysis, rat anti-PD-L1 mAb 4G12 was used as the control protein.To produce the canine chimeric anti-PD-L1 mAb 4G12 (c4G12), the expression vector, pDC6-c4G12, was prepared as described in the supplementary methods. For stable expression of chimeric mAb, pDC6-c4G12 was introduced into DHFR–deficient CHO-DG44 cells  using Lipofectamine LTX Reagent (Life Technologies) following the manufacturer’s instructions. Stable producer cells were selected in CD OptiCHO medium (Life Technologies) supplemented with 2 mM GlutaMAX I (Life Technologies). Cloning of expressing cells was performed following a standard protocol of limiting dilution. Gene amplification was performed by the DHFR/MTX method using CD OptiCHO medium supplemented with 2 mM GlutaMAX I and 60 nM MTX  in combination with further cell cloning. The culture supernatant of the expressing cells was harvested on day 14 of a shaking culture  and the recombinant protein was expressed according to the manufacturer’s instructions. cCD80-Ig was purified from culture supernatant harvested on days 2 and 7 using Ab-Capcher Extra (Protenova), and the buffer was exchanged with PBS using PD-MidiTrap G-25 . The concentration of cCD80-Ig was measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).For the preparation of recombinant canine CD80 protein fused to rabbit IgG Fc (cCD80-Ig), the nucleotide sequence encoding the putative extracellular region of cCD80 was amplified by polymerase chain reaction (PCR) using cDNA obtained from beagle PBMCs stimulated with phorbol 12-myristate 13-acetate  and ionomycin  and the gene-specific primers (5′-CGC GGA TAT CAT GGA TTA CAC AGC GAA GTG-3′ and 5′-CGG GGT ACC CCA GAG CTG TTG CTG GTT AT-3′). The amplicon was then cloned into the multicloning site of the pCXN-2.1–rabbit IgG Fc vector p value of less than 0.05 was considered statistically significant.To evaluate the ability of anti-PD-L1 antibody to block PD-1/PD-L1 or CD80/PD-L1 binding, a blocking assay was conducted on a microwell plate using recombinant canine PD-1, PD-L1 and CD80 proteins  as described in the supplementary methods. Briefly, a flat bottom microwell plate was coated with cPD-1-Ig or cCD80-Ig and blocked with PBS containing 1% bovine serum albumin and 0.05% Tween20. Biotinylated cPD-L1-Ig was preincubated with rat anti-PD-L1 mAb 4G12 or the canine chimeric mAb c4G12 at various concentrations  for 30 min at 37 °C and added to the plate. cPD-L1-Ig binding was detected using avidin-horseradish peroxidase and tetramethylbenzidine substrate. Relative optic density (% OD) was calculated from the OD in comparison with that of control without antibody (0 μg/mL). Rat IgG (Sigma-Aldrich) or dog IgG  was used as a negative control. Statistical analysis was performed by Tukey’s test among groups treated with 10 μg/mL of antibody. A 15, except for the use of gene-specific primers containing the C-terminal 6 × polyhistidine-tag–encoding sequence (5′-CGC GGC TAG CAT GAG AAT GTT TAG TGT CTT-3′ and 5′-CGC GGA TAT CTT AAT GGT GAT GGT GAT GGT GAG TCC TCT CAC TTG CTG G-3′). The amplicon was then cloned into the multicloning site of the pCXN-2.1 vector 38 using NheI and EcoRV restriction enzyme sites. The resulting expression vector, pCXN2.1–cPD-L1-His, was introduced into Expi293F cells (Life Technologies) and the recombinant protein was expressed according to the manufacturer’s instructions. cPD-L1-His was purified from culture supernatant harvested on days 2 and 7 using TALON Metal Affinity Resin , and the buffer was exchanged with PBS by ultrafiltration using Amicon Ultra-4 Ultracel-3 (Merck Millipore). The concentration of cPD-L1-His was measured with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).To assess binding avidity of 4G12, c4G12, cPD-1-Ig and cCD80-Ig to canine PD-L1, SPR analysis was performed using polyhistidine-tagged canine PD-L1 protein (cPD-L1-His). For the preparation of cPD-L1-His, the nucleotide sequence of the putative extracellular region of canine PD-L1 was amplified by PCR as described previously39.SPR analysis was performed using Biacore ×100 system with a CM5 sensor chip and His Capture Kit . Anti-histidine antibody was immobilised on the sensor chip by amine coupling following the manufacturer’s instructions. cPD-L1-His was captured on the sensor chip to obtain approximately 35 response units (RU) to analyse 4G12 or c4G12 binding, or approximately 400 RU to analyse cPD-1-Ig or cCD80-Ig binding. HBS-EP+  was used for both the running and the dilution buffer. Control run responses containing buffer only were subtracted to obtain specific binding responses. The kinetic constants of 4G12 or c4G12 were determined by fitting with the 1:1 kinetic binding model, and that of cPD-1-Ig or cCD80-Ig was determined by fitting with the two state reaction model because a conformational change of PD-L1 upon binding with PD-1 and CD80 has been suggested15, in the presence of 5 μg/mL staphylococcal enterotoxin B (Sigma-Aldrich) and 20 μg/mL c4G12. Dog IgG (Jackson ImmunoResearch Laboratories) was used as a control antibody. For cytokine assays, the culture supernatant was harvested on day 3 and the concentrations of IL-2 and IFN-γ were measured by Duoset ELISA canine IL-2 and IFN-γ , respectively, according to the manufacturer’s instructions. For the proliferation assay, incorporation of nucleotide analogue was evaluated using a Click-iT Plus EdU flow cytometry assay kit (Life Technologies). The thymidine analogue, EdU, was added to the culture medium at a final concentration of 10 μM on day 2, and cells were harvested after additional incubation for 2 h. Cells were stained with optimal concentrations of anti-canine CD4 antibody (R&D systems) conjugated with PerCP-Cy5.5 using a Lightning-Link Antibody Labeling Kit  and anti-canine CD8 antibody  labelled with R-PE using a Zenon Mouse IgG2a Labeling Kit (Thermo Fisher Scientific). After washing, incorporated EdU was labelled with Alexa Fluor 647 following the manufacturer’s instructions. Fluorescence of the cells was analysed with a FACS Verse flow cytometer . For the analysis, the lymphocyte population was gated using forward scatter and side scatter, and EdU incorporation in CD4+ cells or CD8+ cells was evaluated within the lymphocyte population. A Wilcoxon signed rank-sum test was performed to compare data obtained from the same individuals. The result was considered statistically significant if the p value was less than 0.05.PBMCs were prepared from healthy beagles and cultured as described previouslyAntibodies used in clinical studies must be highly purified to maximise the therapeutic potency and to avoid possible side effects. To reduce impurities and aggregates, culture supernatant containing c4G12 was obtained as described above and purified in a series of steps. First, c4G12 was purified from the supernatant by affinity chromatography using a HiScale 26/20 column packed with MabSelect SuRe LX . Additional purification by hydroxyapatite chromatography was performed using a BioScale CHT20-I prepacked column (Bio-Rad), and fractions containing aggregates were further purified by anion exchange chromatography using a HiScreen Q-Sepharose HP prepacked column . Throughout the purification steps, an ÄKTA avant 150 chromatography system  was used. The buffer was exchanged with PBS using a Vivaspin20 concentrator with 50 kDa molecular weight cut of membrane  and stored at 4 °C until use.15. Dogs treated with previous therapies such as surgery, radiation therapy or chemotherapy were included, while dogs with severe systemic illness or autoimmune disease were excluded from the study. c4G12 was diluted in 50 or 100 mL of saline depending on the body weight of dog  and administrated at 2 or 5 mg/kg, every 2 weeks, intravenously using a syringe pump over 1 h. During the treatment period, the dogs were monitored with physical examination, complete blood count and serum chemistry  at least every 2 weeks. The tumour size was measured by a caliper and recorded every 2 weeks if measurable lesions were present on the body surface. At baseline (within 2 weeks prior to the first c4G12 administration) and every 6 weeks during treatment, computed tomography (CT) was performed to evaluate the tumour burden and metastases. For dogs with OMM, TNM-based staging was performed using the World Health Organization staging scheme40 at the time of study enrolment by baseline CT and fine-needle aspiration/cytology of lymph nodes when required. The tumour burden was calculated as the sum of the longest diameters of all target measurable lesions. Tumours ≥10 mm in the longest diameter were considered as measurable lesions. A maximum of five target lesions were chosen from measurable lesions at baseline, with a maximum of two lesions per organ. Tumour response to c4G12 treatment was defined as follows: complete response (CR), disappearance of all detectable tumour; partial response (PR), at least 30% reduction in the sum of the long diameters of target lesions; stable disease (SD), less than 20% increase or 30% reduction in the sum of diameters for at least 6 weeks and progressive disease (PD), at least 20% increase in the sum of diameters. The longest diameters of new measurable lesions  were included in the sum. Unequivocal progression of nontarget lesions was considered PD . If concomitant radiation therapy was given for local control, the lesions targeted by radiation were excluded from the response evaluation to avoid overestimation. No potentially immunosuppressive medications  were given concomitantly with the chimeric mAb treatment. Nonimmunosuppressive drugs, including NSAIDs, antibiotics, anodynes and antitussives were allowed for use during the study where clinically necessary. Adverse events were graded and recorded according to the veterinary cooperative oncology group – common terminology criteria for adverse events (VCOG-CTCAE) v 1.144.Before starting the clinical trial, the safety of c4G12 was confirmed in an experimental dog  by inoculating c4G12 intravenously at 2 mg/kg, every 2 weeks, 3 times in total. No allergic reactions or acute toxicity was found during 2 months of observation period. Therefore, to evaluate the clinical efficacy of c4G12, a pilot clinical study was conducted at Veterinary Teaching Hospital, Hokkaido University. The study period was from March 2016 to January 2017. Dogs with OMM or undifferentiated sarcoma were enrolled in the study if the expression of PD-L1 was confirmed in the cancer cells. The expression of PD-L1 in the primary tumour sections, obtained by surgical excision at prior surgery or biopsy, was assessed by immunohistochemistry as described elsewhere using anti-bovine PD-L1 mAbn = 15, control group). Among the dogs enrolled in the pilot clinical study, all four dogs with stage IV disease had pulmonary metastases . Kaplan-Meier method was used to estimate the median survival of control group and treatment group. Kaplan-Meier curves were generated using statistical analysis software JMP12 , and a log-rank test was used to compere the survival patterns. A p value of less than 0.05 was considered statistically significant.Survival from the confirmation of pulmonary metastasis to death was retrospectively investigated in dogs with OMM treated at the Veterinary Teaching Hospital, Hokkaido University from 2013 to 2016. The dogs treated with standard therapy, such as definitive/palliative surgery, definitive/palliative radiation therapy or chemotherapy using cyclophosphamide/carboplatin at any time from diagnosis to death were included (Supplementary Information