Background. Recent reports indicate that matrix metalloproteinase-7 (MMP-7) and CC-chemokine ligand 18 (CCL18) are potential disease markers of idiopathic pulmonary fibrosis (IPF). The objective of this study was to perform direct comparisons of these two biomarkers with three well-investigated serum markers of IPF, Krebs von den Lungen-6 (KL-6), surfactant protein-A (SP-A), and SP-D. Methods. The serum levels of MMP-7, CCL18, KL-6, SP-A, and SP-D were evaluated in 65 patients with IPF, 31 patients with bacterial pneumonia, and 101 healthy controls. The prognostic performance of these five biomarkers was evaluated in patients with IPF. Results. The serum levels of MMP-7, KL-6, and SP-D in patients with IPF were significantly elevated compared to those in patients with bacterial pneumonia and in the healthy controls. Multivariate survival analysis showed that serum MMP-7 and KL-6 levels were independent predictors in IPF patients. Moreover, elevated levels of both KL-6 and MMP-7 were associated with poorer survival rates in IPF patients, and the combination of both markers provided the best risk discrimination using the C statistic. Conclusions. The present results indicated that MMP-7 and KL-6 were promising prognostic markers of IPF, and the combination of the two markers might improve survival prediction in patients with IPF. Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease of unknown etiology with a median survival of 2-3 years from the time of diagnosis. At present, high-resolution computed tomography (HRCT) is an essential component of the diagnosis of IPF . SurgicaKrebs von den Lungen-6 (KL-6), surfactant protein-A (SP-A), and SP-D are type II pneumocyte-derived molecules which have been investigated by our group and other investigators for their usefulness as serum biomarkers of IPF –6. TheseIn addition to the abovementioned type II pneumocyte-derived biomarkers, recent reports indicate that matrix metalloproteinase-7 (MMP-7) and CC-chemokine ligand 18 (CCL18) are potential diagnostic and prognostic markers of IPF. MMP-7 has been shown to be upregulated in the lungs in IPF, particularly in alveolar macrophages and hyperplastic epithelial cells . BronchoThe aim of this study was to perform direct comparisons of the abovementioned five serum biomarkers as disease markers for IPF. We evaluated the serum levels of the five biomarkers in patients with IPF and control subjects, which consisted of patients with bacterial pneumonia (BP) and healthy controls (HC), and determined the relative values of these biomarkers in discriminating IPF patients from control subjects. Moreover, we examined independent predictive values of serum markers for survival of patients with IPF and tested their additive predictive ability compared with clinical information using the C statistic.Sixty-five patients with IPF, 31 patients with BP, and 101 HC were included in the present study. IPF was diagnosed by clinical features, laboratory findings, chest HRCT, and/or surgical lung biopsy, according to the ATS/ERS/JRS/ALAT statement [1), were performed according to the ATS/ERS recommendation [LCO) was measured by the single-breath method in IPF patients, and lung function measurements were performed at diagnosis in these patients. Reference values were obtained from Japanese reference values for spirometry and DLCO, and the percentages of predicted normal values were calculated.Spirometric measurements, including vital capacity (VC) and forced expiratory volume in 1 second  kits . Serum KL-6 levels were measured by sandwich-type electrochemiluminescence immunoassay (ECLIA) using a Picolumi 8220 Analyzer , as previously described .The results were expressed as the mean ± SD. Demographic characteristics and the levels of serum biomarkers were compared between the subject groups using Bonferroni's test. The levels of serum biomarkers were further analyzed by receiver operating characteristic (ROC) curves to determine the cut-off levels that resulted in the optimal diagnostic accuracy for each marker between the 65 patients with IPF and the 132 control subjects, including BP and HC. The use of these cut-off levels allowed the calculation of sensitivity, specificity, diagnostic accuracy, and likelihood ratio of the five biomarkers for separating the IPF patients from the control subjects. A likelihood ratio above 10 indicates strong diagnostic evidence .p value was < 0.05.In the survival analysis of the IPF patients, another ROC curve analysis was conducted to find an optimal cut-off level for the prediction of 5-year survival. The 5-year mortality between two groups was compared using the Kaplan-Meier method and the log rank test. Univariate and multivariate Cox proportional hazards model was used to identify predictors of 5-year survival in IPF patients. Martingale residuals plots were employed to check for assumptions of the proportional hazards and the linearity of each biomarker. The plots were visually evaluated with the help of locally weighted regression scatterplot smoothing . SubsequThe mean age of the IPF patients was 69.3 years, and the patients with IPF were significantly older than the HC. The mean pack-years of smoking were 37.6 in IPF patients, which was significantly higher than those in the HC. There was no significant difference in age and smoking pack-years between the patients with IPF and those with BP. In the lung function analysis, the mean % VC in patients with IPF was significantly lower than that in the HC .Baseline serum levels of the five biomarkers in patients with IPF were significantly higher than those in the HC. Moreover, serum levels of MMP-7, KL-6, and SP-D in patients with IPF were significantly elevated compared with those in patients with BP. However, there was no significant difference in the serum levels of CCL18 and SP-A between patients with IPF and patients with BP. Moreover, serum levels of MMP-7, CCL18, SP-A, and SP-D were significantly elevated in patients with BP compared with the HC .http://dx.doi.org/10.1155/2016/4759040). Subsequently, we evaluated the discriminative power of each biomarker to differentiate between IPF and BP and between IPF and HC, separately. Only KL-6 showed a high discriminatory ability whereas SP-A and CCL18 were poor indicators for discriminating IPF from BP (Table S1(a) and Figure S2(a)). On the other hand, all five biomarkers were useful to distinguish patients with IPF from the HC (Table S1(b) and Figure S2(b)).ROC curve analysis was used to evaluate the discriminating capability of the five serum biomarkers to differentiate IPF patients from control subjects . Cut-offThe median followup period in IPF was 31.0  months. To find an optimal cut-off level that could discriminate survivors from nonsurvivors, another ROC curve was drawn (figure not shown). Survival in IPF patients using biomarker levels above or below the cut-off level was estimated using the Kaplan-Meier method. Survival was significantly different between higher and lower levels of MMP-7, CCL18, and KL-6 .p = 0.0336) and KL-6  were shown to be independent predictors for 5-year mortality , 1.074; ortality . The fitAs shown in In the present study, we directly compared the diagnostic and prognostic value of five serum biomarkers—MMP-7, CCL18, KL-6, SP-A, and SP-D—in patients with IPF and control subjects. Multivariate Cox analysis showed that serum levels of MMP-7 and KL-6 were independent predictors of prognosis in IPF patients. In addition, IPF patients with elevated levels of both KL-6 and MMP-7 had worse survival rates, and the combination of the two markers with the baseline covariates provided the highest C index. These findings indicated that both MMP-7 and KL-6 were promising prognostic markers of IPF, and a combination of the two markers might improve the survival prediction in patients with IPF. Additionally, we showed that MMP-7 and KL-6 could clearly differentiate IPF patients from patients with bacterial pneumonia and healthy controls, suggesting their potential as diagnostic biomarkers.In this study, MMP-7 and KL-6 were independent predictors of prognosis in patients with IPF, which was consistent with the results of previous reports , 21. MorThe present results indicate that MMP-7 could be a promising diagnostic marker of IPF. The ROC curve analysis showed the excellent discriminative capability of MMP-7, as indicated by the high area under the curve (AUC) value >0.90) and likelihood ratio >10). It should be noted that the ability of a biomarker to discriminate IPF from BP does not necessarily indicate that the biomarker is sufficient, by itself, for diagnosing IPF, although low false positive rates in BP would be an important feature for diagnostic markers for ILDs as shown in our previous study [0 and lik. It shouOur study also demonstrated that serum levels of CCL18 had moderate discriminatory ability for differentiating IPF patients from the HC; however, CCL18 was found to be a poor indicator for distinguishing IPF from BP. An in vitro study reported that CCL18 had a chemotactic effect on lung fibroblasts and stimulated collagen production . HoweverThe present study had several limitations. First, this study is a retrospective review of patients with IPF prospectively recruited from one tertiary hospital, and only Japanese participants who agreed to join this study were included. Therefore, our results may not be generalized to all patients with IPF. Second, there are distinctions in age and smoking histories between patients with IPF and HC. Third, control groups in this study consisted of only patients with BP, an acute lung disease, and healthy subjects; patients with chronic lung diseases, especially other ILDs, were not included in the control group. Therefore, we did not fully evaluate the diagnostic utility of the serum biomarkers. It should be noted that no clinically useful biomarker for distinguishing IPF from other ILDs has been found. We have previously reported that serum levels of KL-6 were also elevated in patients with hypersensitivity pneumonia (HP) . Ishii eOur results showed that both MMP-7 and KL-6 might be a useful prognostic marker of IPF, and a combination of the two markers may improve survival prediction in patients with IPF. Additionally, we showed that MMP-7 and KL-6 could differentiate IPF patients from patients with bacterial pneumonia and healthy controls. These results indicate that the measurement of serum levels of KL-6 and/or MMP-7 could potentially support the diagnosis of IPF and would be useful for identifying vulnerable patients especially when the two markers are used in combination. Further large-scale investigation would be warranted to confirm this finding and to find the best method to use this combination of biomarkers of IPF.Supplementary table 1 and supplementary figure 2 shows the diagnostic value and cut-off levels of the five biomarkers to discriminate patients with IPF from those with bacterial pneumonia and healthy subjects, separately.Supplementary figure 1 shows the ROC curve analysis in serum levels of KL-6, MMP-7 and the combination of serum levels of KL-6 and MMP-7 to distinguish IPF patients from patients with bacterial pneumonia and healthy controls. Supplementary figure 3 shows the Kaplan-Meier analysis of the IPF patients using biomarker levels.Supplementary figure 4 shows the Martingale residuals plots employed to check for assumptions of the Cox proportional hazards model.


Most human microbiota studies focus on bacteria inhabiting body surfaces, but these surfaces also are home to large populations of viruses. Many are bacteriophages, and their role in driving bacterial diversity is difficult to decipher without the use of in vitro ecosystems that can reproduce human microbial communities.We used chemostat culture systems known to harbor diverse fecal bacteria to decipher whether these cultures also are home to phage communities. We found that there are vast viral communities inhabiting these ecosystems, with estimated concentrations similar to those found in human feces. The viral communities are composed entirely of bacteriophages and likely contain both temperate and lytic phages based on their similarities to other known phages. We examined the cultured phage communities at five separate time points over 24 days and found that they were highly individual-specific, suggesting that much of the subject-specificity found in human viromes also is captured by this culture-based system. A high proportion of the community membership is conserved over time, but the cultured communities maintain more similarity with other intra-subject cultures than they do to human feces. In four of the five subjects, estimated viral diversity between fecal and cultured communities was highly similar.Because the diversity of phages in these cultured fecal communities have similarities to those found in humans, we believe these communities can serve as valuable ecosystems to help uncover the role of phages in human microbial communities.The online version of this article (doi:10.1186/s40168-015-0124-3) contains supplementary material, which is available to authorized users. Human body surfaces are inhabited by diverse viral communities, and the majority of those identifiable viruses are bacteriophages , 2. The Bacteriophages possess the capacity to alter microbial communities by either lysing their hosts or providing phenotypic advantages to recipient bacteria –15. PhagStudies of microbiota can be limited due to restrictions imposed by working with human subjects . For exaModel microbial systems must be reproducible, highly stable, and must retain similar levels of diversity to the inocula from which they were derived , 28. Int9 FSPs in feces and 1.4 × 109 FSPs in the chemostat cultures for all subjects studied. The presence of such high densities of FSPs in both sample types strongly suggested the presence of substantial viral communities.We recruited five human subjects through the University of Guelph and sampled their feces. Donors #1, #2, and #10 were a co-habiting family unit of father, mother, and child, respectively, while donors #8 and #9 were unrelated. Each fecal sample was homogenized and processed immediately into a chemostat vessel, and the remainder of the feces was stored until processing of the viromes could take place. Chemostat vessels were operated under conditions designed to mimic the human distal colon . Culture−5) and found that all were free of 16S rRNA gene homologues. We also used BLASTN to search the viromes for homologues against a human reference genome, and some similar sequences  were identified and removed prior to further analysis. These data suggest that these chemostat and fecal viromes were relatively free of contaminating cellular nucleic acids.We isolated and processed viruses from both feces and chemostat cultures utilizing sequential filtration followed by cesium chloride density gradient centrifugation according to our previously described protocols . We sequp = 0.01)   to determine the relative proportion of our viromes that had significant sequence similarities. We utilized the numbers of reads assigned to each contig to identify the proportions of reads belonging to contigs with significant sequence similarities in the NR database. We found that in all subjects combined, 94.9 ± 1.7 % of the reads belonged to contigs that had similar sequences in the NR database  from donor #1 Fig.  and 1981We also analyzed the cultured viral communities using the MG-RAST Server  and cateWe identified significant sequence similarities to each assembled viral contig by BLASTX analysis against the NCBI non-redundant database to decipher which viral genes had similarities in the chemostat cultures. The vast majority of the contigs had similarities to hypothetical phage proteins, proteins involved in replication/integration, restriction/modification enzymes, or tail fibers . Similar results were found for fecal communities, where 25 % of the viruses sampled across subjects had significant sequence similarities compared with only 13 % when comparing between cultured communities and fecal communities (p = 0.141).We previously demonstrated that phages in the mouth are highly persistent members of human oral microbial communities . We utilp ≤ 0.05) trend observed, where the viruses in each of the cultured communities were significantly individual-specific.We created assemblies from all contigs in each donor to determine the relative proportions of viral contigs that were conserved in each donor over time. In donor #1, 53.3 ± 4.0 % of the contigs were conserved amongst the chemostat cultures over time compared to 25.9 ± 3.0 % conserved between the chemostat cultures and the feces  based on the beta diversity between the fecal and cultured viromes to determine if there were patterns of variation specific to each subject and sample type. Each of the cultured viromes reflected the subject from which they were derived Fig. . While tWe developed tools for characterizing viral communities to discern whether richness and diversity were conserved between fecal and cultured communities. The technique, termed the Homologous Virus Diversity Index (HVDI), is primarily based upon the Shannon Index  and is uWe next used the HVDI to perform rarefaction analysis to determine whether the viruses in the viromes had been adequately sampled and as a measure of whether the richness of viruses differed substantially between fecal and cultured communities. In this case, we calculated the HVDI based on the Chao1 index  because We next compared the results of the HVDI using the Shannon Index to investigate whether the diversity of the viral component of cultured communities was similar to that from the feces in each subject. We found that for all subjects, viral diversity in the cultured communities changed as a function of time. For example, in donor #2, viral diversity generally increased from day 4 to day 24, while viral community diversity generally decreased in donor #9. For donors #1, #2, and #10, the diversity present in cultured communities on day 24 was highly similar to that in the feces of each subject Fig. . For donHuman body surfaces are home to large populations of viruses, whose role as members of human microbial communities is not well understood. In some environments, viral communities have been shown to be involved in driving bacterial diversity, yet no such data exists for human viral communities. We know that many of the phages that populate these communities are highly persistent and carry numerous pathogenic gene functions such as antibiotic resistance, which could help to shape bacterial community membership and its response to certain perturbations. Measuring their effects in humans is not without its challenges, which include the need to use drugs such as antibiotics, the need to reduce confounders such as diet variability, and the need to improve compliance with study protocols. The development of ecosystems that can approximate the dynamic interactions between phage communities and their cellular hosts can greatly reduce the reliance on human subjects in the characterization of human microbial communities.Each virome in this study was subjected to MDA amplification due to the small amounts of viral DNA recovered from each donor. MDA amplification is known to introduce biases into sequence data , and it Different bacteriophage families often have different lifestyles. Because phages can be significant drivers of diversity in different ecosystems , 39, 50,Because of the high diversity in human phage communities and the varying relationships between host and phages, the relative abundances of phages do not necessarily reflect those of their host bacteria. This phenomenon was demonstrated in a prior study of human oral viral communities  and in aOur prior studies of human viral communities have demonstrated that diversity in these communities is generally overestimated . In thatOne of the major goals of this work was to examine how closely cultured communities might approximate human indigenous phage communities. Identifying numerous FSPs and many reconstructed phages in the cultured communities Figs.  suggest By establishing phage communities that have some similarities to those found on human body surfaces, several important questions can be addressed. These questions include the following: (1) what is the role of phages in driving the diversity of the bacteria in human microbial communities?, (2) how do perturbations such as antibiotics impact human phage communities?, (3) how does the sharing of phage communities between individuals impact microbial community membership?, and (4) what are the dynamics of most phages as members of human microbial communities? While there are quantifiable differences between the phage communities present in feces and cultured communities, there also are many similarities. The relative number of FSPs in both sample types are similar, the profiles of beta diversity strongly suggest a conservation of some phage community members across fecal and cultured communities, and the relative levels of phage diversity between communities in some subjects were highly similar. By establishing cultured phage communities, we can begin to understand the role and contributions of phages to human microbial communities.Human subject recruitment and enrollment in this study was approved by The Research Ethics Board of the University of Guelph REB#13AP008 and 10JL002. Each subject signed a written informed consent confirming their willingness to participate in this study.Five healthy donors provided fecal samples: donor #1 , donor #2 , donor #8 , donor #9 , and donor #10 . Donors #1, #2, and #10 were a co-habiting family unit of father, mother, and child, respectively. Donors #8 and #9 were unrelated. None of the donors had a recent history of antibiotic treatment for 9 months prior to sampling. Each donor provided at least 5 g of fresh fecal samples for the chemostat cultures. Donor #10 provided a sample in the home environment which was immediately wrapped in plastic cling wrap to exclude air and then frozen at −20 °C for overnight transportation to the lab. The remaining donors provided fresh samples.2, 5 % CO2, 5 % H2) upon receipt at the lab; for the fresh samples, this was within 30 min of collection. The sample from donor #10 was allowed to thaw in the chamber. A modified Infors Multifors system was used to run chemostat cultures modeling the human distal colon environment, as described by McDonald et al. [w/v) slurry was prepared from each donor by homogenizing feces in pre-reduced growth medium using a stomacher. For every 1 L of medium, the following components were included in the growth medium: peptone water, 2 g; yeast extract, 2 g, NaHCO3, 2 g; CaCl2, 0.01 g; pectin (from citrus), 2 g; xylan (from beechwood), 2 g; arabinogalactan, 2 g; starch , 5 g; casein, 3 g; inulin (from Dahlia tubers), 1 g; bile salts, 0.5 g NaCl, 0.1 g; L-cysteine HCl, 0.5 g; K2HPO4, 0.04 g; KH2PO4, 0.04 g; MgSO4, 0.01 g; hemin, 0.005 g; and menadione, 0.001 g . Growth media was stored at 4 °C until use for a maximum of 2 weeks. The fecal slurry in growth medium was then centrifuged to remove large particles [−1 with constant sparging of O2 free N2 gas to maintain positive pressure and anaerobiosis. Each chemostat vessel was sampled daily by aseptically removing 4 mL of culture directly from the vessel contents, and all samples were archived at −80 °C. Aliquots of the original fecal samples also were archived at −80 °C for virome processing.All samples were placed immediately into an anaerobic chamber  of the supernatant from each donor was resuspended in 190 μL of 0.02-μm filtered PBS and their counts per milliliter determined by epifluorescence microscopy [Fecal viromes were prepared by diluting 0.4 g of feces in 4 mL of SM buffer. The fecal samples were vortexed vigorously for 40 min to separate viral particles, spun at 4000×croscopy . Chemostcroscopy . Only thcroscopy  was reta−5) against the Ribosomal Database Project 16S rRNA genes database [ftp://ftp.ncbi.nlm.nih.gov/genomes/H_sapiens/. Any reads with significant sequence similarities to human sequences were removed prior to further analysis. Length and GC content variation amongst contigs was assessed using Box and Whiskers plots created using Microsoft Excel 2007 . All reads were assembled using CLC Genomics Workbench 8.01 based on 98 % identity with a minimum of 50 % read overlap, which were more stringent than criteria developed to discriminate between highly related viruses [www.phantome.org; ftp://ftp.ncbi.nih.gov/genomes/Viruses/) was performed using CLC Genomics Workbench 8.01  and were mapped using 98 % identity over a minimum of 50 % of the read length. For each donor, we also utilized a separate technique for assembly by constructing global assemblies from all contigs from all time points using 98 % identity over a minimum of 50 % overlap. Viral contigs were analyzed using FGenesV  for ORF prediction and individual ORFs analyzed using BLASTP analysis against the NCBI non-redundant database . If the best hit was to a gene with no known function, lower level hits were used for the annotation as long as they had known putative function and still met the E-value cutoff (10−5).Due to the error rate of semiconductor sequencing , we trimdatabase  and the  viruses . The ass viruses . We also viruses  and IDBA viruses  in the c−5. Specific viral sequences were identified by parsing BLASTX results for known viral genes including replication, structural, transposition, restriction/modification, hypothetical, and other genes previously found in viruses for which the E-value was at least 10−5. Each individual virome contig was annotated using this technique; however, if the best hit for any portion of the contig was to a gene with no known function, lower level hits were used as long as they had known function and still met the E-value cutoff. The annotation data were compiled by the number of reads used to assemble each contig for each subject and used to determine the relative proportions of contigs that contained viral sequences. The phyla of the BLASTX best hits for each annotated contig were used to create profiles in each donor and sample type. We utilized the average coverage in the assemblies of each contig to determine the relative abundance profiles of different phyla to compensate for viruses that may be more abundant than others. This technique prevented reads involved in the assembly of the same virus contigs from being assigned to different putative host phyla based on different BLASTX similarities. Determination of the relative abundances of virus families was determined by BLASTX analysis of the SEED database using MG-RAST [Contigs were annotated using BLASTX against the NCBI NR database with an E-value cutoff value of 10 MG-RAST .−10). Principal coordinates analysis (PCOA) was performed on virome contigs with Bray Curtis distances using Qiime [Analysis of shared sequence similarities present in each virome was performed by creating custom BLAST databases for each virome, comparing each database with all other viromes using BLASTN analysis . We amplified the bacterial 16S rRNA gene V1-V2 hypervariable region using the forward primer 8F fused with the Ion Torrent Adaptor A sequence and one of 23 unique 10 base pair barcodes and reverse primer 357R fused with the Ion Torrent Adaptor P1 from the each donor and sample type . Resultip value was computed as the fraction of times the simulated statistic for each exceeded the observed statistic. For analysis of fecal versus cultured viromes, a randomly chosen donor from the cultured virome group was compared with a randomly selected donor from the fecal group to determine the null distribution of the fraction of shared contigs. We then estimated the fraction of shared sequences with significant similarities from randomly chosen donors within the cultured virome group and compared with the empirical null distribution from simulated inter-group values. Intra-subject comparisons were excluded from this analysis. We estimated the p value based on the fraction of times the intra-group statistic exceeded that for the null statistic.To assess whether virome contigs had significant overlap within or between donors and sample types, we performed a permutation test based on resampling . We simulated the distribution of the fraction of virome sequences with significant similarities from two different sample types within a donor. For each set, we computed the summed fraction of sequences with significant similarities using 1000 random contigs between different donors, and from these computed an empirical null distribution of our statistic of interest (the fraction of shared similar sequences). The simulated statistics within each donor were referred to the null distribution of inter-donor comparisons, and the ftp://ftp.ncbi.nlm.nih.gov/genomes/Viruses/) and Phantome viral databases (www.phantome.org). First, a random set of viruses was chosen from amongst the databases for each virome, and viromes were constructed that contained 10 viruses, 50 viruses, 100 viruses, 500 viruses, and 1000 viruses. We constructed 10 different viromes at each level of different viral genotypes. The reads in each virome were of mean length 200 bp and were chosen randomly across the genomes of each virus. The shortest reads were 150 bp, and the longest reads were 250 bp in each constructed virome. Each virome also was constructed to meet specific evenness requirements, where an evenness of close to 1 would indicate a population consisting of different viruses all of similar relative abundances, and an evenness close to 0 would indicate a population where the relative abundance of a few viruses are much greater than others. We utilized evenness as criteria to construct the viromes because higher evenness values would prove more difficult for the assembly process. For each constructed virome, we created contig spectra by counting the number of reads assigned to each different virus in the virome. The contig spectra then were used as surrogates for population structures in determination of the Shannon Index [−20) over 50 % of the length of the shorter contig were assigned to the same viral genotype. The spectra from each individual contig that were assigned to the same genotype were added to a corrected contig spectrum for each subject and those spectra used as inputs for the Shannon Index [To measure alpha diversity in the viral communities, we utilized a technique termed the Homologous Virus Diversity Index . The tecon Index . The acton Index  to calcuon Index , which uAll sequences including viromes and 16S rRNA genes are available for download in the MG-RAST database (metagenomics.anl.gov/) under the project “Chemostat,” or project #10563.


Programmed cell death-ligand 1(PD-L1) was expressed in various malignancies, and interaction with its receptor programmed cell death 1 (PD-1) often contributed to immune evasion of tumor cells. In this study, we explored the expression of PD-L1 and its correlation with clinical outcomes in gliomas.Clinicopathological data of 229 patients with gliomas was collected. PD-L1 expression was assessed by tissue-microarray-based immunohistochemistry. Over 5% of tumor cells with cytoplasm or membrane staining was defined as PD-L1 positive expression. The associations of clinicopathological features with overall survival (OS) and disease-free survival (DFS) were analyzed by univariate analysis and multivariate analysis was further performed by Cox regression model.PD-L1 positive expression was observed in 51.1% gliomas patients and no significant association was verified between PD-L1 expression and pathological grade in 229 gliomas patients. However, PD-L1 expression rate was 49.2%, 53.7% and 68.8% for grade II, III and IV in 161 patients with those ≥ 12 months of OS, respectively. Although no significant discrepancies was displayed, there was a certain degree of differences between PD-L1 expression and pathological grade . Univariate analysis showed that PD-L1 expression was significantly associated with poor OS in the patients with long-time survival or follow up (OS ≥ 12 months) (P = 0.018), especially in patients with grade IV (P = 0.019). Multivariate analysis revealed that a strong tendency towards statistical significance was found between PD-L1 expression and poor OS (P = 0.081).In gliomas patients with long-time survival or follow up, PD-L1 positive expression could indicate the poor prognosis and it is possible that immunotherapy targeting PD-L1 pathway needed to be determined in the further study. Gliomas are the most common brain tumor and they were categorized into low-grade gliomas , and high-grade gliomas  according to the following criteria including cell density, cell atypia, mitoses and presence or absence of necrosis . The conIn the past decades, molecular targeted therapies are beneficial to improve the prognosis of gliomas, such as bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF) , nimotuzProgrammed cell death-1 (PD-1)/Program death-ligand 1(PD-L1) pathway is a classic immune checkpoint of promising immunotherapeutic strategies. PD-L1 could help tumor cells immune evasion in combination with immunomodulatory properties . BlockagThe clinicopathological features of 229 patients were demonstrated in Table In the study, the correlation of PD-L1 expression with the clinicopathological features was demonstrated in Table Univariate analysis showed that PD-L1 expression was not significantly correlated with OS  was achieved for the retrospective analysis. Part of the enrolled patients was dead. Therefore, it was not deemed to be necessary by the ethics committee, and all samples were anonymous.In the study, we collected information for clinicopathological features and survival time in patients with gliomas, 229 samples were achieved in Sun Yat-Sen University Cancer Center from January 2000 to August 2008. The screening of specimens was suitable for the following conditions: gliomas was diagnosed by 2008 World Health Organization classification of central nervous system tumors; The complete clinicopathological data included age, gender, tumor site, histological subtype, pathological grade; neither previous malignancies nor an secondary tumor; without neoadjuvant chemotherapy (IC), radiotherapy (RT) and chemoradiotherapy (CRT) before surgical resection, follow-up regularly. The Institute Research Medical Ethics Committee of Sun Yat-Sen University granted approval for this study.Tissue microarrays were constructed in accordance with a previously described method . In brieFormalin-fixed, paraffin-embedded gliomas samples were cut into 4-μm thick sequential sections and processed for IHC according to the previously described protocol . The TMAThe expression status of PD-L1 was evaluated by microscopic observation of stained TMA slides. The staining of cytoplasm or membranes was deemed to be positive for PD-L1 expression in over 5% tumor cells . PD-L1 eStatistical analysis was performed with SPSS software, version 16.0 . The correlation between PD-L1 expression and clinicopathological features was assessed by chi-square test. Kaplan–Meier method was used to evaluate the relationship between survival time and clinicopathological parameters. Survival time included overall survival  and disease-free survival . The Cox proportional hazards regression model was used to define independent prognostic biomarkers that inversely impacted OS and DFS. A two-tailed P-value less than 0.05 was served as statistically significant.


Daphnia pulex is an especially attractive system for interrogating the gain and loss of introns in natural populations. In this study, we used a comparative population-genomic approach to identify and characterize 90 recently gained introns in this species. Molecular clock analyses indicate that these introns arose between 3.9 × 105 and 1.45 × 104 years ago, with a spike in intron proliferation approximately 5.2 × 104 to 1.22 × 105 years ago. Parallel gains at homologous positions contribute to 47.8% (43/90) of discovered new introns. A disproportionally large number of new introns were found in historically isolated populations in Oregon. Nonetheless, derived, intron-bearing alleles were also identified in a wide range of geographic locations, suggesting intron gain and, to a lesser degree, intron loss are important sources of genetic variation in natural populations of Daphnia. A majority (55/90 or 61.1%) of the identified neointrons have associated internal direct repeats with lengths and compositions that are unlikely to occur by chance, suggesting repeated bouts of staggered double-strand breaks (DSBs) during their evolution. Accordingly, internal, staggered DSBs may contribute to a passive trend toward increased length and sequence diversity in nascent introns.As one of the few known species in an active phase of intron proliferation, the microcrustacean  Giardia lamblia  by the spliceosomal machinery after transcription . Among e lamblia  to an av lamblia . Althoug lamblia .Because introns are often found at the same positions in numerous orthologous genes of widely diverged eukaryotic species , it is lMost attempts to estimate rates of intron gain and loss rely on matrices of intron presence or absence in alignments of highly conserved regions of homologous genes , 2007b. The restrictions of such studies to a small subset of highly conserved genes sampled from distantly related species, to unambiguously aligned regions, and usually to just one sequence per species raise concerns with respect to the generality of the conclusions drawn from such studies. In addition, deep phylogenetic comparisons are of limited use for testing hypotheses regarding the molecular mechanisms of intron gain and loss because the molecular evidence of such events has generally been erased by accumulated mutations. Furthermore, broad phylogenetic comparisons of shared intron positions usually rely on assumptions of character-state irreversibility  repaired by nonhomologous end joining (NHEJ)   and gaing (NHEJ) . Other cg (NHEJ) . Additiog (NHEJ) , reinserg (NHEJ) , or prolg (NHEJ) . Moreovee common .D. pulex, expanding on our previous work (D. pulex clones and a subsequent polymerase chain reaction (PCR)-based survey of 84 additional D. pulex clones sampled from across North America. Because D. pulex harbors substantial nucleotide diversity, it was further possible to use gene phylogenies to assess the polarity of intron gains and losses. New insights regarding the age of derived, intron-bearing alleles and likely molecular events in the early evolution of introns after their initial formation are discussed in the context of our results.Although natural populations harboring intron presence/absence polymorphisms are ideal systems for identifying the molecular footprints of recent intron gains, to date, only a few studies have reported such polymorphisms . Here, wous work . These nD. pulex clones were sequenced and analyzed for intron presence/absence polymorphisms .Complete genomes of nine orphisms . Each of project . AnotherDNA used for high-throughput sequencing was extracted from >200 individuals of each clone using a Qiagen DNeasy tissue kit  following manufacturer’s instruction. DNA libraries were prepared and sequenced by the Beijing Genome Institute. Each clone was shotgun sequenced using an Illumina genome analyzer, yielding ≥20× coverage per site. Raw paired-end reads, each ∼75 bp in length, were de novo assembled using SOAP . More thA), a Perl script was used to generate 100-bp hybrid sequences comprising 50-bp portions of the exons flanking each intron in the TCO genome. These hybrid sequences were used to BLAST against contigs of sequenced genomes of the other eight clones of D. pulex. This strategy allowed us to find intron absence alleles in non-TCO clones of D. pulex. The same strategy was used for intron presence/absence comparisons between TCO and the D. magna Finnish clone.Two main strategies were used to search for intron presence/absence polymorphisms among newly assembled genomes. In Strategy I A, a PerlB), each exon in TCO was used to BLAST against newly assembled contigs of the other eight D. pulex clones to search for exonic insertions that are present in non-TCO clones but absent in TCO. Sequences of top hits were aligned using ClustalX  from conserved flanking protein-coding regions around the gain–loss polymorphisms to generate an ∼1 kb amplicon that contains at least two upstream or downstream introns. Including other nonpolymorphic introns in our targeted PCR amplifications facilitated detection of linked presence–absence polymorphisms, as linked intron loss might simply reflect the presence of processed pseudogenes. In such cases, these sites were excluded from further analysis. The same set of primers was also used to screen cDNA prepared from surveyed clones (see below).Primer pairs were designed using Primer 3 . The QIAprep Spin Miniprep Kit (Qiagen) was used for plasmid purification, and a T7 primer was used to sequence cloned inserts. To avoid PCR and cloning errors, 10–14 cloned products were sequenced per individual for these loci. Additionally, to ensure that cloning-induced errors were not the source of observed polymorphisms, sequences from cloned PCR products were compared with the directly sequenced PCR products .Daphnia individuals. mRNA was extracted using the Qiagen RNeasy Mini Kit (Qiagen). Extracted mRNA was quantified using a NanoDrop 3300 . A total of 1,000 µg of mRNA from each sample was used for cDNA synthesis using the SuperScript III first-strand synthesis kit  following the manufacturer’s instruction. Primers used in genomic DNA-based PCR were applied to synthesize cDNAs using the above-described amplification conditions. Obtained sequences of cDNA fragments were aligned with genomic sequences using ClustalX  part of the alignment of the gained intron sequences is completely conserved, but the remainder is highly divergent. Because introns that fall into these two patterns are homologous for at least part of their lengths, we scored them as a single gain event. To define parallel gains, we apply the following criteria: 1) If insertion sites for two functional introns are within 10 bases of one another, but not exactly the same insertion site, we regard these insertions as parallel gains  A gap in the alignment of gained introns from diverse t-tests were used to assess whether differences in the lengths of exons or the frequencies of their repeats were significant and chi-square tests were used to assess whether differences in base composition were significant and implemented using StatPlus:mac LE.2009 . An additional Python script (RepCheck), described below, was used to compare repetitive DNA content in the above three categories of exonic regions.To assess whether exonic regions surrounding the sites of established introns, singly gained or parallelly gained introns, differed substantively, an original Python script was used to compare their lengths, base composition, repeat content, and repeat frequency. πs) was calculated using DnaSP , and exon regions that are downstream of a derived intron-bearing exon. Sequences from outgroup species were excluded from our calculation. For each exon region, average pairwise sequence diversity at silent sites  Repetitive motifs (excluding microsatellites) within each new intron; 2) repetitive motifs shared by new introns at distinct loci; 3) repetitive motifs shared by exons at distinct loci hosting newly gained introns; and 4) repetitive motifs common to a neointron and one of its adjacent exons.P value of 0.0001 was used to reduce the chance of recovering coincidentally similar sequence segments. However, in the cases of a handful of examined neointrons, these search criteria revealed no significant motifs. For these introns only, an additional lower stringency screen (P ≤ 0.001) was used to recover repetitive elements of potential importance.Short sequence repeats in newly gained introns or adjacent exons were identified using REPFIND . A cut-oSpace_for_2nd_Repeat (stored in the program as Prob_of_2nd_Occurence). The program then compares each repeat identified by REPFIND with each intron in which it occurs. Repeats that have a probability of 0.05 or less of occurring a second time in a given intron are written to a tab-delimited output table. The source code for RepCheck is available from the authors upon request.For the repetitive motifs identified in category one, an addition Python script, called RepCheck (developed for this study), was used to evaluate the probability that any repeat identified by REPFIND would occur twice, by chance, in a particular intron. To evaluate this probability the following quantities were gathered: 1) The frequency of each nucleotide in an intron of interest; 2) the length of the intron of interest ( = Intron_Length); 3) the base composition of the repeat of interest; and 4) the length of the repeat of interest. The quantities are used to assess the probability of a given sequence occurring a second time in a given intron as follows: 1) Multiply together the frequencies of each nucleotide of a repeat in the intron where it is found (the result is stored in the program as Simple_Prob); and 2) determine the available space for a second occurrence of a given repeat in a given intron by taking the length of the intron and subtracting twice the length of the repeat less 1 (the result is stored in the program as Space_for_2nd_Repeat). The probability of the repeat occurring a second time is: 1 − (1 − Simple_Prob)For recovered direct repeats in classes two, three, and four, an additional Python script was used to determine the length, frequency, and base composition of each detected direct repeat. These metrics were subsequently evaluated using StatPlus:mac LE.2009  to assess the following: 1) Their distributions; 2) correlations among them; and 3) significant differences between them. The same methods of analysis were used to evaluate the repetitive content of exonic regions surrounding the sites of single and parallel gains, to assess whether there was a substantive difference between exons in these two categories.D. pulex alleles surveyed for a certain locus following the method in Daphnia species available for each locus to determine the derived state of SNPs with a congruent method. An allele phylogeny for each locus was used to find the most closely related Daphnia species of D. pulex. Once the derived SNPs were identified by this method, the derived allele frequencies of SNPs at silent sites were calculated by dividing the number of derived single nucleotides by the total number of single nucleotides of D. pulex at sampled positions.To compare the allele frequency spectra of gained introns and derived single nucleotide polymorphisms (SNPs), derived allele states of both types were inferred from an outgroup to polarize polymorphisms. Original Python scripts were used to calculate the frequencies for intron-bearing alleles and derived SNPs at silent sites. The frequencies of new introns were calculated by dividing the number of new intron-containing alleles by the total number of µ, for D. pulex is 6 × 10−9 per base per generation at silent sites  (D. pulex clones is 3.4 × 105 years (standard deviation = 8.02 × 104 years). This number is used as a calibration point for molecular clock dating using BEAST.To estimate the approximate age of the most recent common ancestor (MRCA) of th MEGA5  using th. pulex) . The appDaphnia  for a prior distribution, ucld.mean  was set to uniform, and an estimated silent site mutation rate of 6 × 10−8 per base per year was provided; and 6) for Markov chain Monte Carlo, the length of the Markov chain is 1,000,000 generations, echoing state to screen every 10,000 generations, and logging parameters every 200. Thus, the final sample files contained 5,000 trees.BEAST v1.7.2 and associated software applications, BEAUTi v1.7.2, TreeAnnotator v1.7.2, and FigTree v1.3.1, were used to facilitate molecular clock dating to estimate the age of new introns in Daphnia . To prepTreeAnnotator v1.7.2 was used to process the resultant trees. Parameters for this step were set as follows: 1) For burnin, we chose 50 to specify a 1% burnin; 2) for posterior probability limit, we chose 0.5, which summarizes the information for nodes with this posterior probability; and 3) for node heights, we used mean heights. FigTree was used to display the features of summary trees generated by TreeAnnotator. In FigTree, posterior was selected for display of branch labels and node ages for display of node labels.D. pulex populations were calculated in three categories: 1) An average age estimate of all derived intron-bearing alleles that are polymorphic in D. pulex; 2) an average age estimate of new introns that are endemic to Oregon populations only; and 3) an average age estimate of multiple unique introns gained at nearly the same location. Clades used for these calculations met two criteria: 1) They comprise only GIB alleles and 2) they have a posterior probability greater than 0.65 in the final summary tree generated by TreeAnnotator. A histogram of allele ages was generated in R 3.0.1.The results for gained intron-bearing (GIB) alleles that are still segregating in http://blast.ncbi.nlm.nih.gov/Blast.cgi, last accessed June 20, 2014). The full nonredundant/nucleotide (nr/nt) database in GenBank was searched for significant matches. BLAST algorithm parameters were set for “somewhat similar sequences” (BlastN), with other parameters left at default settings. Only hits with an e-value < 0.01 and sequence identify > 75% were deemed to be potential homologs of inserted sequences in Daphnia.BLAST searches were conducted using the sequences of newly gained introns as queries with the nucleotide BLAST program .Through a series of BLAST searches, we obtained orthologous sequences from the genomes of nine recently sequenced 85 genes   AT content in contrast to 55.0 ± 0.2 in adjacent exons of the same gene . Of these, 25 contain in-frame stop codons, an amount that is not significantly different from the expected value, given the overall rate of stop codon-bearing neointrons in our data set. Among the introns that did not contain stop codons, a majority (71%) would still result in frameshift mutations in downstream exons, if unspliced. In total, 88 of the 90 neointrons identified through this study would likely eliminate gene function if they were not spliced from pre-mRNA transcripts .The 90 ame gene . Approxibsupplementary fig. S63, Supplementary Material online), while the other has GG … AG . Rare, alternative splice sites have been observed in other species. For example, in the human genome, 99.1% of the introns have GT … AG boundaries, 0.9% of them have GC … AG boundaries, and 0.1% of them have AT … AC splice site junctions . For example, at locus Dpul_328763 , all outgroup species, all D. pulex clones from Oregon, and 14 non-Oregon clones have a plesiomorphic allele, Dpul_328763(+), that has an intron separating exons 8 and 9. In contrast, a set of geographically diverse populations in a nested clade have an alternative allele, Dpul_328763(−), that lacks this intron and, instead, has a single exon that is homologous to both exons 8 and 9 in Dpul_328763(+) alleles.Combining results of this survey of 84 us study  reveals µ) for D. pulex of 6 × 10−8 mutations per base per year  years, is approximately the same as the MRCA of all D. pulex.Based on the silent site mutation rate  is 1.40 ± 0.09 × 105 (mean ± SE) years. D. pulex populations in Oregon were thought to have experienced one or more historical bottleneck events (5 (mean ± SE) years.Distributions of derived intron-bearing alleles that are still segregating in el gains . Our estk events . Thus, tD. pulex resulted from parallel gain events. The estimate for the average age of allele clades encompassing multiple parallel intron gains at nearly the same location  is 1.13 ± 0.12 × 105 years. Overall, the distribution of ages of the 90 neointrons identified in this study is positively skewed and ranges from 1.45 × 104 to 3.9 × 105 years ago. A pronounced spike in intron accumulation occurred between 5.2 × 104 and 1.22 × 105 years ago .Based on sequence characteristics of these newly gained introns and adjacent exons, observed events of intron gain are associated with initial DSBs. Some appear to have been formed through staggered DSBs, evidenced by direct repeats at opposite ends. Others show evidence of a combination of blunt and staggered DSBs at the same site, although the order of these two events is sometimes unclear (D. pulex. Identified repeats were short segments (ranging from 4 to 17 bases) that occurred more than once (excluding microsatellites) and were discovered through either a high stringency (P ≤ 0.0001) or low stringency (P ≤ 0.001) REPFIND . Repair of staggered DSBs creates direct repeats, leaving a distinctive molecular footprint. However, the presence of some direct repeats is expected to be obscured by subsequent mutations on the timescale of 1/µ (µ being the mutation rate per base per generation).We discovered that a majority of neointrons contain internal repeats caused by staggered DSBs. Probability assessment by RepCheck indicates that 61.1% (55/90) of these internal repeats were unlikely to occur by chance . If internal staggered DSBs were common, recurrent events in introns, we would expect similar percentages of new and old introns to have internal direct repeats. However, a much higher percentage of neointrons have significant internal repeats (61.1%). A possible explanation for these observed differences is that internal DSBs occurred at precisely the time of new intron formation, by complex process that is yet to be explained. Over time, the initial short sequence repeats generated by staggered DSBs will have differentiated from one another through the accumulation of mutations as introns age.The same search strategy was applied to other established introns in the same gene for each surveyed locus. Among 617 examined introns, only 100 (16.2%) have internal repeats that pass the cutoff A; also see examples in supplementary figs. S5 and S11, Supplementary Material online); 2) single staggered DSB followed by sequence insertion ; 3) blunt and staggered DSBs that occurred side-by-side at the same locus, although the order is unclear ; 4) blunt-then-staggered  ; 5) blunt-then-blunt  ; 6) staggered-then-staggered ; and 7) staggered-then-blunt  . The category into which each new intron can most plausibly be assigned is listed in supplementary table S3, Supplementary Material online.New introns identified in this study can be categorized into seven categories, based on the apparent mechanism of their formation : 1) Singfig. 3D. pulex fall into three categories, based on phylogenetic patterns in inferred trees: 1) All D. pulex: an intron-bearing allele is present in all sampled D. pulex clones and no outgroup species; 2) Nested clade: an intron-bearing allele clade is nested within D. pulex, but is absent from other D. pulex clones and all outgroups; and 3) Parallel gain: independently gained, distinct neointrons are present at homologous sites in alleles of separate D. pulex clones, but are not present in any outgroup .1.D. pulex Intron GainsAll Cases of intron gain in Dpul_228023 locus . Here, all outgroup species have an uninterrupted exon 2, while the homologous protein-coding region is interrupted by a single orthologous intron in all sampled D. pulex clones.2.Nested-Clade Intron GainsEight gained introns fall into this category. An example is the D. pulex clones. In 19 of these cases, derived clones form a monophyletic group or groups nested within the larger D. pulex clade. For example, the derived allele Dpul_341016(+) at the Dpul_341016 locus bears a single insertion in a coding region that is homologous to exon 2 at this locus in all other D. pulex clones and outgroup species. Dpul_341016(+) alleles form a clade in the inferred tree and are present in all populations outside of Oregon .3.Parallel Intron Gains In the case of 61 introns in 57 genes, derived introns are present in only a subset of sampled D. pulex exhibit a pattern of parallel intron gain. In total, parallel gains account for 51 derived intron-bearing alleles in sampled populations . All outgroup species and a sizeable number of D. pulex clones have alleles with an uninterrupted exon 11. In three independent events, distinct introns were inserted into this exon in geographically diverse clones of D. pulex. We detected 19 additional genes in D. pulex with patterns similar to that observed at Dpul_308086, although the number of alleles per locus and affected exons varied. In one extreme case, locus Dpul_327924, six different, derived introns were identified in close proximity in alignments of one exon .Twenty genes in ulations . An illuπs, using the Jukes–Cantor correction and averaged across all sampled clones)  results reveal that values of πs of exons targeted by derived introns are significantly higher . These findings indicate that exons that bear derived introns are less conserved than their upstream or downstream neointron-free exons.Average pairwise sequence diversity at silent sites ( clones)  of exonsD. pulex, given that none of the outgroup species have introns at homologous locations. The rest of the neointrons have a frequency ranging from 1.4% to 98.3%. The frequency spectrum of derived intron-bearing alleles is skewed toward lower values  similar to that of derived SNPs at silent sites . One gained intron shares significant sequence identify with a D. pulex ribosomal RNA gene. Three additional significant hits were between Dpul_310811_TCO, Dpul_325592_TCO, and Dpul_210427_TCO, and genomic segments from Dania rerio, Enterocytozoon bieneusi, and Schistosoma mansoni, respectively. Possible explanation of these results would be horizontal transfer events from D. rerio, E. bieneusi, or S. mansoni, or from one of their close relatives for which genome sequences are not available. However, because these neointrons are rich with A and T mononucleotide repeats and e-values ranged from 0.01 to 3.0 × 10−5, microsatellite proliferation could also account for the observed similarity. No other neointrons yielded significant BLAST hits.In our previous study, one neointron shared significant sequence similarity to a sequence . BLAST aDpul_47827 locus reveals that when this intron is spliced out, it includes an 8 bp segment that was previously part of the upstream exonic sequence on its 3′ end . The second mechanism that can cause a length disparity between an inserted segment and the fragment excised during splicing is when an inserted segment has an internal splice site and its unspliced terminus extends the length of its adjacent exon.In a majority of cases, the newly inserted introns have canonical GT … AG splice sites at their termini. Comparison of cDNA sequences and other orthologous exons that do not have recently inserted introns reveals that the entire segment inserted into an interrupted exon was cleanly excised during RNA processing. In contrast, we identified a handful of cases in which the length of an inserted segment and the length of the RNA fragment removed during splicing are not identical. This length disparity may result from either of the two mechanisms. The first entails recruitment of a portion of an adjacent exon to become part of the excised fragment. For example, analysis of cDNA sequences at the Dpul_300453 locus is spliced out, one bp at the 5′ end of the inserted segment is recognized as an extension of the adjacent exon and 4 bp at the 5′ end of the downstream exon are included in the intron that is excised during splicing , resulting in a loss of one amino acid in the encoded protein. Similarly, at Dpul_341016 locus, 6 bp at the 5′ end of the inserted segment serve to extend the adjacent exon and 12 bp at the 5′ end of the downstream exon are excised along with the intron during splicing , resulting in a net loss of two amino acids in the encoded protein. These results indicate that exonic sequence deletion and new intron insertion co-occur at a subset of the surveyed loci.In other cases, both the mechanisms mentioned above may apply. For example, when the derived intron at the P ≤ 0.0001) screen of all neointron sequences of D. pulex using RepFind . There was a strong, positive correlation (P = 1.82 × 10−8) between AT content and the frequency of repeats that are present among several neointrons. High frequency repeats  had an average AT content of 93% and just over two-thirds of these, including the 23 most frequent repeats, had 100% AT content. Individually, high frequency repeats make up between 1.5% and 11.5% of the length of neointrons in which they reside. The abundance and AT richness of high frequency repeats help to explain the high AT content of new introns relative to adjacent exonic regions, which average only 54.7% AT content.This analysis allowed us to investigate if there are any shared motifs among the newly gained introns. A high stringency ( RepFind  identifiP ≤ 0.0001 in RepFind) of exons, which are the host of neointrons, revealed 130 repetitive motifs ranging in length from 4 to 15 bp and in frequency from 2 to 172 occurrences in the collective set of all such exon sequences . AT content of exon-specific repeats (54.6%) is generally similar to the AT content of the exons in which they reside (53.9%). Second, to search for potential sequence motifs that may facilitate single or parallel intron gain, we conducted two separate analyses: a) Exon sequences that exhibit parallel gains were compared with those having a single gain only and b) exons that contain established introns were compared with those bearing neointrons. Comparisons of sequence length, base composition, repeat content, and repeat frequency indicated no significant differences. Hence, we detected no distinguishing features in repeats found in exons that served as sites of neointron colonization once only or more than once. Additionally, none of the surveyed features appear to be unique to exons that have recent insertion of introns when compared with exons with established introns.This analysis allowed us to assess whether: 1) There are any shared motifs among the exons hosting new introns; 2) if so, do any shared motifs facilitate parallel intron gain, and 3) if there is any distinctive sequence features or motifs that distinguish exons colonized by neointrons from exons hosting established introns. Specific motifs in exons might encourage intron colonization or facilitate intron maintenance  . To scresupplementary table S6, Supplementary Material online).In cases where intron formation entailed a staggered DSB, a direct repeat of the terminal base pairs in an adjacent exon is produced at the opposite end of the neointron. Thirty-three direct repeats of this kind were identified in our data set. These repeats range in length from 4 to 12 bp. In the pool of associated intron and exon sequences, these repeats occur between 2 and 87 times and are marginally AT rich (57.3%), but no correlation between repeat AT content and frequency was detected . In sum, although it has been posited that 1) intron gains are rare, 2) intron gains are much less common than intron losses, and 3) intron gains are unlikely to occur more than once at homologous positions, our results show that these notions do not apply equally well to all lineages.Our findings indicate that several longstanding notions concerning the origin of introns differ from the results obtained by this study. First, numerous authors have suggested that intron gains are rare events . In cont Daphnia , we idenlineages , leadinglineages . In contlineages , perhapslineages . Our resDaphnia and previous comparative studies of highly diverged phylogenetic lineages may be attributable, at least in part, to the ages of the lineages investigated. Although the MRCA of sampled D. pulex is estimated to be merely 3.4 × 105 years old, prior studies compared intron presence and absence across widely diverse eukaryotes that may have shared a common ancestor no less than 2.2 × 109 years ago. Several of the neointron-bearing alleles detected in the current analysis are still actively segregating in D. pulex and may not persist in the long run. Although it remains unclear whether patterns that emerge through our analyses apply to lineages of far greater age, at present there is no formal basis for ruling this out. Notably, results similar to those of this study have been observed in other species with actively segregating neointron-bearing alleles  were discovered in these populations of Daphnia. A plausible explanation for this pattern is that the Oregon populations have smaller effective population sizes than their non-Oregon counterparts, and hence experience less effective selection, allowing a greater proportion of mildly deleterious intron-bearing alleles to become fixed . However, in several other cases, the Oregon-allele clade is nested well inside a larger clade comprising all other D. pulex populations . This latter pattern suggests that alleles at these loci originated in populations of D. pulex outside Oregon and were subsequently introduced to Oregon populations of D. pulex. In contrast, none of our inferred allele trees reveal non-Oregon clades nested within a larger Oregon clade. Hence, our results suggest that historical gene flow between Oregon and non-Oregon populations may have been largely or entirely unidirectional and that novel intron-bearing alleles received by Oregon populations are likely to be fixed or lost rapidly, due to drift, resulting in reduced overall genetic variation.In phylogenetic trees of alleles at numerous examined loci, clades comprising all Oregon alleles are sister to all other non-Oregon lineages, suggesting pronounced genetic differentiation between Oregon and non-Oregon populations  (mean ± SE), compared with that from derived SNPs at silent sites  years. In contrast, the average estimated age of derived intron-bearing alleles segregating in D. pulex populations is 1.40 ± 0.09 × 105 (mean ± SE) years, and 1.20 ± 0.9 × 105 (mean ± SE) years for derived intron-bearing alleles in Oregon populations. The frequencies of these alleles at polymorphic loci range from 1.2% to 88.9%. These results suggest that a majority of the derived intron-bearing alleles detected in this study are long lived, having persisted in natural populations of Daphnia for between 50,000 and 300,000 years, but only 6.7% of them appeared to be fixed during that time. We did not detect evidence of a selective advantage associated with new introns, although those that did become fixed may have done so as a result of hitchhiking effects caused by linked, beneficial loci. The distribution of estimated ages of new introns is positively skewed with a spike in the accumulation of recent introns gains in D. pulex between 52,000 and 122,000 years ago (A and B).Eight of the newly gained introns appear to be fixed in ears ago A and B.Arabidopsis reveal a tendency for initial length mutations to engender additional nearby length mutations that may serve to offset deleterious effects of the first indel  might help explain a standing mystery of the origin of intron gains—where do introns come from? Despite a few cases in which the sources of neointrons have been identified  , the souBased on our observation of frequent occurrence of internal repeats, we propose a new perspective to explain the mystery of the early evolution of novel introns. Regardless of the mechanisms of their initial creation, introns may subsequently expand or contract in length, possibly facilitated by repair of ongoing DSBs through NHEJ . AdditioDaphnia genome   or will cause frameshifts in downstream exons if unspliced. Our observation that there is no significant difference in the presence of PTCs between 3n and non-3n introns may reflect that neointrons surveyed in this study are robustly spliced, thus obviating a need for NMD to remove deleterious transcripts. However, a larger number of 3n introns would need to be examined to draw any firm conclusions. In the event that they are not spliced out, retained PTC-free introns might appreciably alter the length and composition of the encoded proteins, which would very likely be strongly deleterious.It has been hypothesized that the permissiveness of spliceosomes to recognize introns with weak splicing site opens the door for intron gain, and that NMD functions as a failsafe mechanism for weak splicing of new introns, especially in 3 introns . This hyintrons) . In contDaphnia genomes. For the various sequences inserted into exons, we propose that there are four potential fates: 1) Newly inserted sequences in exons that do not contain proper splicing signals are unlikely to be spliced and will likely affect gene function adversely, and can be rapidly removed by selection; 2) precise splicing of the entire insert as a neointron; 3) partial splicing that leaves a portion of the inserted sequence in the mRNA transcript; 4) activation of latent splice sites in adjacent exon regions, leading to the splicing of the inserted segment and portions of the adjacent exons. Partial splicing of inserted sequences or recruitment of adjacent exon sequences will cause the truncation or extension of the existing coding regions. If intron gain is a transient phase in the population, such events will accordingly cause elevated sequence diversity in alleles that had gained introns but lost them during the course of intron turnover relative to ancestral intron-free alleles. This might explain the higher sequence diversity (πs) of exons targeted by derived introns compared with adjacent exons. But, it is also possible that coding regions with higher than usual mutation rates may be more vulnerable to DSBs, which could potentially pave the way for new intron formation.Our results suggest that sequence insertions in coding regions are quite common in Supplementary figures S1–S77 and tables S1–S6 are available at Genome Biology and Evolution online .


The development of materials and strategies that can influence stem cell attachment, proliferation, and differentiation towards osteoblasts is of high interest to promote faster healing and reconstructions of large bone defects. Graphene and its derivatives (graphene oxide and reduced graphene oxide) have received increasing attention for biomedical applications as they present remarkable properties such as high surface area, high mechanical strength, and ease of functionalization. These biocompatible carbon-based materials can induce and sustain stem cell growth and differentiation into various lineages. Furthermore, graphene has the ability to promote and enhance osteogenic differentiation making it an interesting material for bone regeneration research. This paper will review the important advances in the ability of graphene and its related forms to induce stem cells differentiation into osteogenic lineages. Bone tissue regeneration is of high interest to promote faster healing and reconstruction of large bone defects created by tumor resection, skeletal abnormalities, fractures, and infection. The development of this field requires the use of substrates that enable cell attachment, proliferation, and differentiation –3. Sever2 carbon atoms and is the thinnest, lightest, and maybe the strongest material known [Graphene is a single atomic sheet of conjugated spal known , 11. Itsal known  making gal known –17. As gal known , 18–23.Since its discovery in 2004 by Nobel laureates Geim and Novoselov , graphenGraphene Physical, Chemical, and Mechanical PropertiesThinnest, strongest, and stiffest imaginable material .Almost transparent , 26.Most stretchable crystal (20% elasticity) .Recording thermal conductivity .Highest current density at room temperature .Completely impermeable .Highest intrinsic mobility (100 times more than in Si) .Conducting electricity in the limit of no electrons .2 g−1) [Large surface area (~2600 m2 g−1) .Longest mean free path at room temperature (micron range) .2 g−1, makes it an attractive platform for anchorage of large amounts of molecules [Due to its unique structure and a pure aromatic carbon system , it has olecules , 33.Graphene-related materials can be classified based on either number of layers  or their chemical modification such as graphene oxide (GO) or reduced GO (rGO) . GO is aGraphene in different forms can be obtained using “top-down” and “bottom-up” methods. The “top-down” approaches include mechanical and/or chemical exfoliation of graphite. The mechanical method, also known as the “Scotch tape” or peel-off technique, allows the detachment of micrometer-sized graphene flakes from a graphite crystal using adhesive tape , 32, 38.4 and H2) are pumped into a reaction chamber  at high temperatures (~1000°C). The high temperature leads to the pyrolysis of precursors and dissociates carbon atoms which then react with the substrate to produce the thin film of graphene [Graphene can also be obtained using “bottom-up” approaches like epitaxial growth and chemical vapor deposition (CVD) , 41. Thegraphene . CVD-grographene , 43–47.−1) arises from the stretching of the C–C bond in graphitic materials whereas the D band (~1340 cm−1) is only activated if disorder or defects are present. The 2D band (2500–2800 cm−1) is present in all types of sp2 carbon materials and is used to determine the number of layers of graphene [The different methods to produce graphene result in materials with different number of layers and/or chemical groups. Raman fingerprints for different groups and number of layers reflect changes in the electron bands and allow unmistakable identification and characterization of graphene by the analysis of three peaks: G, 2D, and D . The G agraphene .Graphene and its derivatives are interesting materials for biomedical applications since carbon is the basis of organic chemistry , 34. HowAnchorage-dependent cells need to adhere to substrates in order to spread, proliferate, and perform their functions , 50, 51.μg/mL) as it accumulates on the cell membrane leading to high levels of oxidative stress [μm can enter mammalian cells initiated by spontaneous penetration of lipid bilayers in a dominant edge-first or corner-first mode. Nonetheless, the uptake of microsheets larger than 5 μm in lateral dimensions is often incomplete [μg/mL), there is an increase in reactive oxygen species production and decrease in metabolic activity. However, at concentrations of 0.01 and 0.1 μg/mL, there is no increase in lactate dehydrogenase, an enzyme released upon membrane damage [Although substrates coated with graphene-based materials are not cytotoxic, the use of the material in solutions might pose hazards to cells and tissues. Cell viability may decrease significantly in solutions with high concentrations of pristine graphene  to minimize oxidation. There was no considerable toxicity after injecting mice with 20 mg/kg with PEGylated graphene as evidenced by histological and hematological analysis after 90 days. In fact, the graphene sheets levels in most organs were very low after three days from the injection. The relatively slow but persistent decrease of the material concentration in the liver and spleen suggests that the clearance of graphene nanosheets from the mouse body happens through both renal and fecal excretions .As graphene-based materials can be functionalized, there is an increased interest in using them for biomedical applications. In fact, the surface functionalization may be an important step for pacifying its strong hydrophobicity that may be associated with toxic effects. Nonetheless, the potential long-term adverse effects of functionalized graphene cannot be neglected . FurtherMajor bone reconstruction represents a major challenge and is a global health problem. Stem cell-based therapy might be a promising solution but it requires the constant development of biocompatible platforms that can promote and enhance cell viability, attachment, migration, and differentiation , 3. SeveGraphene has emerged as a promising material for stem cell research due to its unique mechanical, physical, and chemical properties  20, 24,,24, 61. 2) after 48 hours of incubation. Furthermore, MSCs on graphene assume a spindle shaped morphology whereas those cultured on SiO2 substrates form separate islands of polygonal cells [2 substrate [2, graphene oxide, and polydimethylsiloxane (PDMS) substrates [Cell adhesion, viability, and proliferation rate are directly related to the biocompatibility of the substrate , 6, 51. al cells . As cellal cells  the spinubstrate . Furtherbstrates , 46 but bstrates .2 were coated with CVD graphene, MSCs present high expression of osteocalcin (OCN) as compared to the uncoated materials [Although cell proliferation is not improved by pristine graphene, the material enhances stem cell differentiation towards osteoblastic lineage , 47, 52.aterials . These iaterials . It is katerials . Other faterials , 45, 47.aterials .It is known that the transport phenomena of cytokines, chemokines, and growth factors are drastically different between two- and three-dimensional 3D) microenvironments interfering in signaling transduction, cell-cell communications, and tissue development D microen, 70. Gra2 substrates treated with BMP-2 [β-glycerophosphate [π-π stacking between the aromatic rings in the molecules and the graphene basal plane [Although graphene holds the potential to induce spontaneous osteogenic differentiation of stem cells, this property is significantly enhanced by the use of chemical inductors for osteogenic differentiation , 47, 52.th BMP-2 . These hhosphate , 45. Dexal plane . GO is pal plane , 45. HenAlthough graphene has great benefits for osteogenic differentiations due its excellent physical properties, it can also be chemically modified  or combiPolymers have also been modified with graphene to provide better environments for cell survival and differentiation. The addition of 1 wt% of GO to gelatin-based composite improves significantly the tensile strength, Young's modulus, and energy at break by 84, 65, and 158%, respectively . The addPoly-L-lactide (PLLA) scaffold was modified with CNT and graphene of 1–3% wt by thermal-induced phase separation technique. The scaffold containing 3% wt of graphene enhances the differentiation of BMSC and increases the calcium deposition and formation of collagen type I. This can be attributed to the increase in specific surface area of the scaffold and the surface roughness that can increase adsorption of proteins. Furthermore, graphene provides more contacting surface to cells as compared to the same content of CNT .Graphene can form self-supporting graphene hydrogel (SGH) by the principal of colloidal chemistry due to intrinsic corrugation of graphene and solvation repulsion between neighboring graphene sheets, resulting in a large amount of separated graphene sheets in a collective manner inside SGH , 81. MulDue to the large surface area and delocalized electrons, GO and rGO have the potential to bind and solubilize molecules acting as drug delivery vehicles , 83, 84. in vitro and in vivo [ in vitro osteogenic differentiation of BMSC was higher on Ti/GO combined with BMP-2 than on Ti with BMP-2. Further in vivo experiment demonstrated the efficacy of BMP-2 delivered by Ti or Ti/GO substrate after implantation into mouse calvaria defects. After 8 weeks, Ti/GO implants conjugated with BMP-2 showed extensive bone formation revealed by microcomputed tomography and histological analysis as compared to Ti/BMP-2 substrate [Given that the induction of osteogenic differentiation of stem cells can take several weeks, the sustained release of inducing proteins, such as BMP-2, can accelerate this process. Recently the potential of titanium coated with GO (Ti/GO) has been explored for sustained release of BMP-2 to increase osteogenic differentiation in vivo . Ti subsubstrate .These findings corroborate graphene as a promising material that can increment bioactivity and differentiative potential of candidate materials for bone tissue regeneration. in vivo animal studies are needed to assess biodistribution and their metabolic pathways in tissues and organs to permit future clinical applications.The characteristics of graphene such as large surface area, excellent mechanical properties, and feasibility to be transferred to different substrates among others make it a unique material for stem cell research. Graphene-modified substrates and materials are biocompatible, allow cell adhesion and proliferation, and increase differentiation of stem cells into osteogenic lineage. In addition, it can be easily functionalized to bind biomolecules or elements of choice to induce and control stem cell behavior. Although some challenges remain, the advances obtained by using graphene to induce osteogenesis are exciting. One of these challenges is the lack of thorough and profound understanding of the mechanism and signaling pathways involved in stem cell differentiation stimulated by graphene. Further studies at cellular and subcellular level beyond proof of concept and focusing on underlying mechanism are necessary. Moreover, comparisons of graphene with current known and successful biomaterial and implants must be performed to conclude the benefits of the material. Furthermore,Due to the unique structures and remarkable properties, graphene and its derivatives hold great potential for biomedical applications. Although the research with graphene for bone tissue regeneration is still in early stages of development, the material may have bright future in clinical scenarios.


We demonstrated a method to construct high efficiency saturable absorbers based on the evanescent light field interaction of CVD monolayer graphene deposited on side-polished D-shaped optical fiber. A set of samples was fabricated with two different core-graphene distances (0 and 1 μm), covered with graphene ranging between 10 and 25 mm length. The mode-locking was achieved and the best pulse duration was 256 fs, the shortest pulse reported in the literature with CVD monolayer graphene in EDFL. As result, we find a criterion between the polarization relative extinction ratio in the samples and the pulse duration, which relates the better mode-locking performance with the higher polarization extinction ratio of the samples. This criterion also provides a better understanding of the graphene distributed saturable absorbers and their reproducible performance as optoelectronic devices for optical applications. The graphene is a two-dimensional carbon allotrope nanomaterial consisting of one atom thickness and that has important optical and electronic properties123456789101112131415161719234510111213141516171011121314151617et al. using exfoliated graphene dispersed in a polymer at all-fiber connector tip configuration, which pulses as short as ~800 fs with 3.2 nm bandwidthet al. demonstrated the generation of 756 fs pulses with 5 nm spectrum bandwidth1011121314151617Ultrashort pulses are suitable for numerous applications, which include telecommunications and the study of ultrafast processes3456234567891011121314151617In this work, we present an efficient and reproducible mode-locking performance criterion with seven D-shaped side-polished fiber samples based on CVD monolayer graphene saturable absorbers for EDFL, obtaining pulses duration of 256 fs, which is the shortest pulse reported in the literature with CVD graphene monolayer, for our knowledge. We carried out the characterization process in three steps after the fabrication of the sample by the transference of the graphene to the D-shaped optical fiber: the first step was to verify the quality of the monolayer graphene by Raman spectroscopy. Thereafter, the performance of the monolayer graphene sample as a polarizer was evaluated by the polarization relative extinction ratio between the modes of the light with polarization parallel (TE radiation) and orthogonal (TM radiation) to the graphene’s plane. Finally, we characterized the graphene saturable absorber mode-locking performance relating it to the polarization relative extinction ratio of each sample.−1), G (1592 cm−1) and 2D (2685 cm−1) characteristic bands of the graphene−1, full-width at half-maximun (FWHM), corresponds a monolayer while around 40 cm−1 indicates bilayer grapheneThe samples were analyzed with a Raman Microscopy System  using a 532 nm wavelength laser with an incident power of 2 mW. In TE/PTM), where PTE/PTM is the ratio between the power of the modes TE and TM relative to the graphene’s plane. Without graphene, the insertion loss of the D-shaped optical fiber was 0.60 dB, whereas with graphene the TE and TM losses for this sample were 20 and 6.1 dB respectively, resulting in an extinction ratio of 96%. The The polarization dependent loss of the monolayer graphene D-shaped fiber sample was performed by rotating input power of 754 μW with linear polarization from 0 to 360° degrees. The result is shown in the μm and PMMA thickness variation between 300 and 400 nm. An approaching to analyze this influence and give a support to the relative extinction ratio polarization measurements, were done by simulation, using the COMSOL Multiphysics software,version 5.1. The attenuation calculated by simulation for each orthogonal mode in each sample is summarized in As it can see in The EDFL continuous wave (CW) lasing threshold without the saturable absorber was achieved at a pump power of 15.7 mW. Incorporating the monolayer graphene saturable absorber on the D-shaped fiber, the lasing threshold increased to 20 mW. Non-self-starting mode-locking regime was obtained from 50 to 110 mW pump power, generating soliton-like pulses highly dependent of polarization state. In fact, because of symmetry breaking in the fundamental propagation mode caused by D-shaped optical fiber/graphene configuration and the unequal property of graphene as polarizer and saturable absorber, the mode-locking was activated for high absorption of the TE mode and different polarization states can generate distinct performances in the laser. In The mode-locking performance can be directly related to the sample’s polarization relative extinction ratio as shown in In this work, we present a simple and high performance criterion for D-shaped optical fiber based on monolayer graphene saturable absorber for the generation of ultrashort pulses in EDFL. With this method, it is possible to associate the polarization relative extinction ratio between the TE and TM radiation with the passively mode-locking optimization. In the EDFL, the samples with polarization relative extinction ratio higher than 85% resulted in pulse duration shorter than 300 fs. The shortest pulse duration of 256 fs was obtained at higher polarization relative extinction ratio of 96%, which is the shortest pulse reported in the literature using CVD monolayer graphene. This criterion can be used to evaluate the quality and performance of several optical devices based on transferred graphene in optical waveguides.The polarization relative extinction ratio is an important parameter because it is associated with the saturable absorption mechanism which generate ultrashort pulses. The saturable absorption mechanism is associated with electronic transitions from the valence band to the conduction band due to the interaction of light with graphene at high intensities. Due these transitions, the conduction band levels become fully occupied (Pauli blocking), so the graphene becomes transparent for high intensities. In the experiment and simulation results, high relative polarization extinction ratio implies a high absorption at TE mode, responsible for both saturable absorption in graphene, and ultrashort pulses generation.h) of 0 μm with transferred graphene length (l) of 10, 18.5 and 24 mm; and four samples with core-surface distance of 1 μm and 13, 14, 19 and 25 mm graphene length. The monolayer graphene was transferred from the cooper to the side-polished of the D-shaped optical fiber by using the wet transfer methodThe monolayer graphene was produced by chemical vapor deposition (CVD) grown on cooper and the polished D-shaped optical fibers (Phoenix Photonics) samples were provided, with distances from the fiber core to the surface of 0 and 1 μm and polishing lengths of 17, 25 and 30 mm. A total of seven samples were fabricated, three samples with core-surface distance μS. h = 1 μm; PMMA refractive index 1.49 and 300 nm thickness; fiber core and cladding refractive index 1.4492 and 1.4440 respectively and fiber core diameter 8.2 μm.The graphene was modeled using the boundary condition for the tangential magnetic field How to cite this article: Zapata, J. D. et al. Efficient graphene saturable absorbers on D-shaped optical fiber for ultrashort pulse generation. Sci. Rep.6, 20644; doi: 10.1038/srep20644 (2016).


Graphene/ZnSe film and graphene/CdSe film hybrid photodetectors fabricated by this method also show a high and reproducible performance. The general method is compatible with the conventional planar process, and would be easily standardized and thus pay a way for the photodetector applications.High- and reproducible-performance photodetectors are critical to the development of many technologies, which mainly include one-dimensional (1D) nanostructure based and film based photodetectors. The former suffer from a huge performance variation because the performance is quite sensitive to the synthesis microenvironment of 1D nanostructure. Herein, we show that the graphene/semiconductor film hybrid photodetectors not only possess a high performance but also have a reproducible performance. As a demo, the as-produced graphene/ZnS film hybrid photodetector shows a high responsivity of 1.7 × 10 High- and reproducible-performance photodetectors are critical to the development of many technologies, including communications, sensing, environmental protection, and imaging, etc1235678910121315162 bonded carbon atoms with ultrahigh carrier mobility192021222324252627et al. demonstrated a responsivity of 107 A/W in a hybrid photodetector that consists of graphene covered with a thin film of PbS quantum dotsWe now consider the film based photodetectors. They possess a low performance due to low carrier mobility of the film consisting of nanocrystals. The performance has been greatly enhanced by hybridizing semiconductor films with graphene, a two-dimensional single-layer of sp2/Si substrate by high vacuum method and are supposed to have a fixed amount of photo-generated carriers at different areas. The CVD grown graphene films on Cu foil were subsequently transferred onto semiconductor film29302 V−1 s−1, 7 A/W and a fast response speed of 50 ms, and shows a highly reproducible performance, in terms of narrow distribution of photocurrent (38–65 μA) and response speed (40–60 ms) for 20 devices. Graphene/ZnSe film and graphene/CdSe film hybrid photodetectors fabricated by this method also show a high and reproducible performance. Remarkably, our general method is compatible with the conventional planar process and will be easily standardized.Herein, we show that the graphene/semiconductor film hybrid photodetectors not only possess a high performance but also have a reproducible performance, taking ZnS, ZnSe and CdSe as examples. Semiconductor films were deposited onto SiO2/Si substrate shows that the thickness of ZnS film is observed to be 60 nm, in agreement with the setting value during e-beam evaporation process  obviously increased regardless of the gate bias (Vg) (Rλ) is a key parameter to evaluate the photodetector performanceRλ = ∆I/PSI is the difference between the current under illumination and dark current, namely photocurrent, P is the light power density irradiated on the channel, and S is the area of channel. Vsd) under different light power, with the back gate of Vg = 0 V. The photocurrent increases with the increase of Vsd and light power, while the calculated responsivity increases with the increase of Vsd yet decreases with the increase of the light power. It is consistent with the photodetector behavior reported before21Rλ value of our fabricated graphene/ZnS film photodetector is up to 1.7 × 107 A/W at an incident power of about 10 μW/cm2, indicating a high responsivity for our graphene/ZnS film hybrid photodetector. The responsivity can be expected to be further enhanced under light illumination with a lower power121516To fabricate graphene/ZnS film based field effect transistors, 10/50 nm Cr/Au electrodes were deposited by thermal evaporation method with standard photolithography and lift-off processes. After 365 nm light was focused on the channel with size of 5 × 5 μm inset in , the pho2126ias (Vg) . The spe2 V−1 s−1  and response speed (40–60 ms) for 20 devices. The method universality is confirmed by the realization of graphene/ZnSe film and graphene/CdSe film hybrid photodetectors with a high and reproducible performance. Our general method developed in this work is compatible with the conventional planar process, and would be easily standardized and pay a way for the detector applications.In summary, the combination of graphene films possessing an ultrahigh mobility and uniform conductivity with semiconductor films having a fixed amount of photo-generated carriers allows us to achieve graphene/semiconductor film hybrid photodetectors with a high and reproducible performance, based on that film based detector has a reproducible performance because it is collective one of the nanocrystals in film. As a demo, the produced graphene/ZnS film hybrid photodetectors shows a high responsivity of 1.7 × 102 was utilized as substrate for the semiconductor film deposition. The vacuum remained 2 × 10−4 Pa during the whole deposition process. Before graphene transfer, the semiconductor film on Si/SiO2 substrate was annealed for 30 min at 300 °C under low pressure with 10 sccm H2 and 20 sccm Ar, enabling the film adhered to the substrate as it was placed in water to pick up the graphene films.ZnS and ZnSe film with the thickness of 60 nm was deposited by e-beam evaporation, and 60 nm thick CdSe film was produced by thermal evaporation. ZnS, ZnSe and CdSe crystals (99.99% purity) with size of mm scale were used as targets. After the standard cleaning, the Si wafer with 300 nm SiO2 and 20 sccm Ar. This method enables the crackles transfer of graphene.The details of graphene growth and transfer can be found in our previous work312/Si, photolithography was used to fabricate transistors with channel lengths of 5 μm and widths 5 μm. For the performance reproducibility investigation of the graphene/ZnS, graphene/ZnSe and graphene/CdSe hybrids, 20 devices with channel length of 20 μm and width of 500 μm were fabricated with shadow mask. The doped Si substrate served as a global back gate. 10/50 nm Cr/Au used as the electrical contact metal for the source and drain electrodes were deposited by a high-vacuum thermal evaporation system.After graphene was transferred onto ZnS, ZnSe and CdSe film on 300 nm SiOSEM images were taken with a Hitachi SU8020 scanning electron microscope operated at 1 kV. XRD patterns of the samples were recorded with an X-ray diffractometer (X’ Pert Pro MPD) with λ = 1.54056 Å. Raman spectroscopy was performed on the samples with a confocal microprobe Raman spectrometer (Renishaw). UV-vis absorption (UV-3600) was conducted on the samples after transfer onto quartz substrate. The electrical and photoconductive properties of the graphene/ZnS film, graphene/ZnSe film and graphene/CdSe film hybrid devices were measured by a semiconductor parameter analyzer system (Keithley 2636B) at room temperature, using light emitting diodes with the corresponding wavelength of 365 nm, 460 nm and 620 nm as the incident light.How to cite this article: Huang, F. et al. High- and Reproducible-Performance Graphene/II-VI Semiconductor Film Hybrid Photodetectors. Sci. Rep.6, 28943; doi: 10.1038/srep28943 (2016).