Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer associated with poor prognosis and additional therapeutic strategies must be implemented to optimize ESCC treatment. Meanwhile, the important biologic role and potential prognostic and therapeutic implications of a tumors immunologic microenvironment (IM) have been recognized in various cancers.PD-L1 copy number status via fluorescence in-situ hybridization.In order to investigate the contexture and the prognostic relevance of the IM in ESCC, we immunohistochemically evaluated the extent of overall/intraepithelial TILs (CD3+/CD8+) and of PD-1 / PD-L1 expression in a cohort of 125 therapy-naive ESCCs, additionally assessing p = 0.019; PD-L1: p = 0.028), disease specific- (DSS)  and disease free survival (DFS) . CD3ihigh- and PD-L1high cases were significantly associated with one another (p < 0.001). Subgrouping of ESCC revealed decreased OS (p = 0.031), DSS (p = 0.012) and DFS (p < 0.001) for CD3ilow/PD-L1low cancers.High intraepithelial CD3+ TILs (CD3ihigh) and high PD-L1 expression on tumor cells (PD-L1high) were each significantly associated with improved overall- (OS) (CD3+: Our data not only associate CD3ihigh- and PD-L1high ESCC with a beneficial outcome, but also demonstrate PD-L1high- and CD3ihigh status to be closely intertwined. Furthermore, our study demarcates a prognostically unfavorable, “non-immunoreactive” CD3ilow / PD-L1low ESCC-subgroup, potentially forming the basis for an immune-based stratification of ESCC. Esophageal squamous cell carcinoma (ESCC) is a substantial cause of cancer-related death worldwide, accounting for approximately 80% of esophageal cancers –6. ESCC The recognition of the immunologic microenvironment (IM) of a malignant tumor as a potentially powerful biomarker of prognostic and therapeutic relevance has led to a resurgence of immune based therapy strategies in solid cancers. Firstly, the immunologic tumor-host relationship appears to be of substantial importance for the carcinogenic process , 9 and eHowever, in ESCC, data regarding its IM are limited and the prognostic value of PD-L1 expression is still a matter of debate, as some studies associate PD-L1 expression with a rather favorable , 22 progPD-L1 copy number status was evaluated through Fluorescence in situ Hybridization (FISH).In order to investigate the contexture and possible clinicopathological implications of the IM including the PD-1 / PD-L1 axis in ESCC, we immunohistochemically analyzed the extent and distribution of overall and intraepithelial CD3+ / CD8+ TILs as well as PD-1 / PD-L1 expression in a tumor series of 125 primary resected, therapy-naive ESCCs. Additionally, the tumors (1) the specific composition of the IM in ESCC, (2) the identification of possible associations of overall and intraepithelial TILs with specific clinicopathological and survival parameters, (3) the predictive value of PD-L1 immunohistochemistry in ESCC and (4) the relationship of the PD-1 / PD-L1 axis to the IM, particularly focusing on potential immunologic ESCC subgroups based on their extent of T-cell infiltration and PD-L1 positivity.The questions we addressed focused on 95 of the 125 ESCC patients in our tumor series were male (76%), 30 were female (24%). Mean age at diagnosis was 60 years (range: 39-83). Rather locally advanced cancers  and early stage carcinomas  were roughly evenly distributed, synchronous lymph node metastases (pN+) were detectable in 57 cases (46%) and four cases showed synchronous distant metastases (pM1: 3%). According to the WHO classification of the digestive system , most ESIntraepithelial CD3+ TIL (CD3i) count was heterogeneous showing a wide range from 0-70/100 (median: 12) tumor cells (TC). 44/125 ESCCs exceeded the 66. percentile, harboring >20 CD3is , while the remaining cancers had <20 CD3is and were therefore classified as CD3ilow. 50 tumors (40%) showed diffuse CD3i distribution. Intraepithelial CD8+ TIL (CD8i) count fluctuated from 0-70/100 TCs (median: 6). 38 tumors clustered within the upper third and had >13 CD8is . Diffuse CD8is were detectable in 40 (32%) cases. Intraepithelial PD-1+ TIL (PD1i) count varied from 0-25/100 TCs (median: 3) and 41 cases had a high PD1i count of >6 PD1is, exceeding the 66. percentile  . Overall density of CD8+ TILs varied from 1-50% , whereas overall PD1+ TILs ranged from 0-60%  .Immunohistochemically, at least partial membranous PD-L1 staining was detectable in 71% of ESCCs , showing a range from 0-90% positive tumor cells. 38/125 (30%) cases were classified as PD-L1high as their amount of PD-L1 positive TCs exceeded the 66. percentile (>10% positive TCs). Weak (34%), intermediate (31%) and strong (35%) staining intensities were almost evenly distributed among the PD-L1 positive cases.PD-L1+ TILs were detectable in 109/125 cases (87%) and their density within the whole tumor area varied from 0-60%   and PD-L1 deletions in 10/125 (8%) ESCCs. The remaining cases displayed PD-L1 disomy  or polysomy  (Table Assessment of the %) Table .Slightly modifying the recently proposed immune-based cancer classification system by Teng et al. , ESCCs wAs highlighted in Table PD-L1 copy number status were not associated with clinicopathologic variables. Overall density of TILs across the tumor area (including TCs and tumor stroma) showed no significant associations with clinicopathologic features.CD3ihigh tumors were significantly associated with lower pT stage (p=0.01), lower UICC stage (p=0.001) and tumor free local lymph nodes (p=0.001). Cases showing a diffuse distribution of CD3is were also more frequent in lower pT stage (p=0.02) and lacked nodal involvement more often (p=0.03). PD1ihigh tumors showed lower pT stage (p=0.015) and frequently lacked nodal involvement (p=0.02). CD8ihigh and PD-L1high status, PD-L1 expression in TILs and UICC stage , pT , pN  and pM  stadium were all significantly associated with survival parameters, while conventional histopathologic grade only displayed a comparatively weak association with OS (p=0.039), but not with DSS or DFS. Age and sex played no role in survival prediction  revealed improved survival parameters for CD3ihigh tumors , diffusely distributed CD3is , diffusely distributed CD8is  and PD-L1high ESCCs . Details on multivariate survival analyses are given in Table As demonstrated in Table Additional ESCC subgrouping based on T-cell infiltration CD3i status) and PD-L1 status revealed the CD3ilow / PD-L1low subgroup to be significantly associated with reduced OS (p=0.031), DSS (p=0.012) and DFS (p=0.001) compared to the other subgroups. Mean OS  of patients with CD3ilow/PD-L1low ESCC was 46.8 months  compared to 76.9 months  for patients with CD3ilow / PD-L1high tumors, 86.4 months  for patients with CD3ihigh / PD-L1high cancers and 95.4 months  for patients with CD3ihigh / PD-L1low ESCCs  than in squamous cell carcinoma of the oral cavity (19%) [Data on expression rates and prognostic value of PD-L1 immunohistochemistry in ESCC is rather scarce and partially conflicting, as some studies associate PD-L1 positive ESCC with poor patient outcome , 25, 32,ty (19%) , indicatty (19%) . FurtherBesides in ESCC, the association of immunohistochemical PD-L1 expression to a beneficial outcome has been observed in several entities like colorectal carcinoma , non-smaTaking our analyses one step further, we stratified our ESCC cohort into four immunogenic subgroups based on their PD-L1 / CD3i status. Interestingly, our subgrouping approach, which represents a slightly adjusted version of the TIL/PD-L1 based cancer classification proposed by Teng et al. , unmaskeOur work has some limitations: (1) our investigations were performed on TMA-basis, although we examined a substantial number of tumor cores deriving from the diverse tumor compartments . Furthermore (2), only resection specimens (3) without postoperative checkpoint blockade were investigated. As our work is retrospective in nature, our data need to be validated in larger, prospective ESCC cohorts as well as on biopsy material. Furthermore, the suitability of PD-L1 IHC, intraepithelial CD3+ TILs and the subsequent immunologic ESCC subgroups as a potential rationale for immune checkpoint therapy regimens clearly needs to be investigated by subsequent clinical studies in appropriate patient cohorts.Summarizing, we investigated the contexture of ESCCs IM and demonstrate increased intraepithelial CD3+ TILs and high PD-L1 expression on tumor cells to be independent predictors of a beneficial clinical outcome in ESCC. This study not only highlights the comparatively frequent expression of PD-L1 in this tumor entity and underlines the close association of PD-L1 positive ESCCs with increased numbers of intraepithelial T-lymphocytes, but also identifies a subset of non-immunogenic ESCCs with a distinct clinical course, potentially forming the basis for an immune-based stratification in this tumor entity.Our tumor series comprised 125 ESCC patients, who underwent surgical resection between 1994 and 2007 at Klinikum Rechts der Isar of the Technical University of Munich, Germany and at the University Hospital Heidelberg, Germany. All tumors were chemo-/radiation-naive at the time of resection and none of the patients received immune checkpoint inhibitors or underwent other immune therapy regimens. Only tissue from primary tumors was investigated. Hematoxylin-Eosin (H&E) stained sections were initially reviewed by two pathologists , who confirmed the diagnosis of ESCC. Grading and staging of ESCC at the time of diagnosis was performed according to the current World Health Organization Classification of Tumors of the Digestive System  and the Formalin-fixed paraffin-embedded (FFPE) tumor samples from the central and apical tumor region as well as from the invasive margin were assembled into a tissue microarray (TMA) using a Tissue Microarrayer  with a core size of 0.6 mm. All samples of a respective tumor region  were extracted from areas harboring a high tumor/stroma ratio. Mean tumor cell content per core was 79.3%, ranging from 40% - 99 %. Obvious inflammatory hotspots (such as lymph follicles or areas of ulceration) were avoided. Considering tumor heterogeneity, a minimum of 3 and (where feasible) up to 6 tumor cores were taken from the primary tumors in areas previously marked by five pathologists .IHC was performed on 2 μm sections from each TMA using a PD-L1 primary antibody : SP-263; dilution (d): 1:100), a PD-1 primary antibody  and primary antibodies against CD3  and CD8 , using an automated immunostainer with an iVIEW DAB detection kit .Scoring of TILs  was performed jointly by two pathologists  blinded to clinicopathological outcome. In general, TILs were separately evaluated in every core of the TMA and an average score resulting from all cores  was assigned as the final TIL count for the respective case. The analysis of TIL- subpopulations was performed in two ways: (1) Scoring of TILs was performed in the tumor region of the respective core showing the highest density of the particular TIL subpopulation on low power magnification (4x). Within this region, the amount of intraepithelial CD3+ TILs (CD3i), intraepithelial CD8+ TILs (CD8i) and intraepithelial PD-1+ TILs (PD1i) was scored manually by counting the quantity of the respective lymphocyte subpopulation within tumor cell clusters of 100 tumor cells using high power magnification (40x). (2) In analogy to previous TIL-scoring approaches , 47 overPD-L1 expression in TCs and in TILs was jointly determined by two pathologists  and was evaluated separately in every TMA core before a final score resulting from all investigated cores  was assigned. In TCs, the absolute percentage of positive cells was determined. Only membranous staining patterns were scored as positive. The intensity of PD-L1 staining was scored using a 4-tiered grading system established in a recent study regarding the efficacy of the used PD-L1 SP263 antibody , includiFollowing the general scoring process, the respective data were quantified regarding their respective percentile value and patients were stratified into three subgroups: lower (below 33. percentile), intermediate (33.-66. percentile) and upper third (exceeding 66. percentile). For further analyses, cases within the upper third were classified as “high”, whereas all cases ranging within the intermediate or the lower third were classified as “low”.CD274, PDCD1LG2/CEP9 dual color probe and PD-L1 copy number status (CNS) was evaluated as described previously counting at least 20 tumor cell nuclei per tumor core [PD-L1 amplification was defined as PD-L1/CEP9 ratio ≥2.0, with high level amplification defined as ≥4.0 and low level amplification defined as ≥2.0 and ≤4.0. Polysomy 9 was defined as average PD-L1 copy number >3 signals/cell. PD-L1 deletion was defined as PD-L1/CEP9 ratio <0.8.FFPE tissue sections (thickness 2μm) of TMAs were used for dual-color FISH analysis using a SPEC mor core . PD-L1 a2 test and Fisher's exact test. Associations of immunologic factors with each other were calculated with Spearman's rank order correlation. Survival probabilities were plotted using the Kaplan-Meier method and a log-rank test was used to probe the significance of differences in survival probabilities. Multivariate survival analysis was performed utilizing the Cox proportional hazard model. All significances were two-sided, p-values ≤0.05 were considered significant.Statistical analyses were performed using SPSS 23 . Correlations between immunologic characteristics and clinicopathological parameters were calculated using Χ


XBP1 mRNA contain short introns that are not removed by the spliceosome. There have been some efforts to identify additional short introns, but little is known about how many short introns are processed from mRNAs. Here, we report an approach to identify RNA short introns from RNA-Seq data, discriminating against small genomic deletions. We identify hundreds of short introns conserved among multiple human cell lines. These short introns are often alternatively spliced and are found in a variety of RNAs–both mRNAs and lncRNAs. Short intron splicing efficiency is increased by secondary structure, and we detect both canonical and non-canonical short introns. In many cases, splicing of these short introns from mRNAs is predicted to alter the reading frame and change protein output. Our findings imply that standard gene prediction models which often assume a lower limit for intron size fail to predict short introns effectively. We conclude that short introns are abundant in the human transcriptome, and short intron splicing represents an added layer to mRNA regulation.Canonical pre-mRNA splicing requires snRNPs and associated splicing factors to excise conserved intronic sequences, with a minimum intron length required for efficient splicing. Non-canonical splicing–intron excision without the spliceosome–has been documented; most notably, some tRNAs and the  Most pre-mRNA introns are excised by the major or minor spliceosome . BothDuring RNA-Seq, introns are predicted most effectively by checking for conserved intron sequences. Introns are initially indicated when sequencing reads align discontinuously with the reference genome; the gaps in sequencing alignments, or putative introns, are then further investigated. If the sequence of the gap begins and ends with GU-AG, AU-AC, or closely-related sequences, and the length is greater than a user-defined minimum (often 50 nucleotides), then the intron is defined. The flanking sequences are considered exons ,15. Thesin vivo [S. cerevisiae, tRNA ligase completes splicing of the HAC1 (homologous to ATF/CREB 1) mRNA [But non-canonical introns do exist. Some spliceosomal introns have degenerate splice site sequences. RNA-Seq alignment algorithms commonly deal with the most abundant degeneracy, GC-AG rather than GU-AG, but other splice site sequences are tolerated in vivo . More imin vivo  (yeast Sin vivo ). In addin vivo –23). IRE 1) mRNA , and in  1) mRNA ). In bot 1) mRNA –22). TheWith RNA-Seq datasets, there is a wealth of sequencing data that can be used for short intron identification, but conventional sequencing analysis overlooks this intron class. Recently, one group has attempted to identify short introns ,26. TheyXBP1 mRNA, newly-identified short introns are not constitutively spliced, but rather are alternatively spliced. Taken together, our findings extend the intron family to include hundreds of additional short introns.Here, we have identified short introns more comprehensively. We report the identification of hundreds of human short introns that range in length from 10 to 70 bases. Many short introns are found in mRNA open-reading frames and would be expected to alter protein output when spliced. A majority of short introns maintains major-class splice site consensus sequences, but many do not. We identify these short introns not only in mRNAs, but also in long non-coding RNAs (lncRNAs). Short introns are enriched for secondary structure. As is true for the short intron in XBP1 mRNA short introns are shorter than this cutoff, we extended the normal search parameters to include introns with a predicted length down to 10 nucleotides. We chose this lower limit for two reasons: genomic deletions are infrequent at lengths greater than 10 nucleotides [Intron prediction algorithms commonly exclude any predicted intron less than 50 nucleotides in length . Since bleotides ,17, and leotides –22,25–27i.e. splice sites that lack either GU-AG or AU-AC consensus sequences. STAR alignment produced many predicted short introns which we filtered extensively to remove predicted introns with little experimental support as well as likely genomic deletions. Briefly, we used between-replicate reproducibility, removed likely genomic deletions, and checked for conservation in multiple cell lines  which we first compared to other published intron datasets. First, we assessed the average lengths for all predicted introns to ensure that our search parameters returned results consistent with the work of others. Next, we focused on identifying short introns. Since the splicing machinery has been documented to require a minimum intron length ,13, we qAt this point, many predicted short introns could have been observed due to spontaneous genomic deletion in the various cell lines, and some additional short introns, in many cases, had little sequencing read support. We therefore further filtered the predicted short intron sequences.For each cell line, the ENCODE data contained two biological replicates making it possible to do pairwise comparisons. Normally, a false discovery rate could be calculated comparing predicted introns with a reference set, but there is no reference set for short introns. Therefore, we employed the non-parametric Irreproducible Discovery Rate (npIDR) approach ,34. WithWe next attempted to remove spontaneous genomic deletions from our predicted intron pool. Tissue culture cell lines contain various genomic abnormalities, including aneuploidies, genomic insertions, and genomic deletions ,17. Of tSpontaneous genomic deletions are common when repetitive DNA sequences are replicated by DNA polymerase . For exaMany of the predicted short introns remaining in our pool were poorly conserved between cell lines. To further narrow down the number of predicted short introns, we took advantage of the fact that the search algorithm predicted many longer introns that were unannotated in the reference genome. We therefore compared these longer, unannotated introns against our pool of short introns. For each longer intron, both annotated and unannotated, we first used the npIDR cutoffs established above to remove predicted introns with little sequencing support. We then queried the number of samples  containing the remaining longer introns. Results were tabulated and converted to a percentage of the total shown in . Once eaXBP1 mRNA (XBP1s) has been analyzed for splicing efficiency [XBP1s splicing approaches 80–90% [XBP1s would be spliced inefficiently. In all but one cell line, we detected sequencing reads for exon-exon junctions resulting from XBP1s splicing  to 0% (in the K562 cell line). When aligned sequencing reads were plotted versus genomic coordinates for the Mcf7 cell line, we observed a dip in sequencing reads at the position of XBP1s . The complete results of our analyses are contained in Since each short intron was alternatively spliced in at least one cell line, we next queried features of the short introns that might contribute to splicing efficiency. Surprisingly, splicing efficiency was not governed by whether the short intron contained canonical splice site sequences. Shown in The conserved sequences within a major-class intron recruit components of the splicing machinery, such as U1 snRNP and U2 snRNP ,9. DurinTo examine short introns in more detail, we selected the 600 short introns with the greatest sequencing support, ranging from 50 unique sequencing reads to 37,514 unique reads. These short introns fell into three main categories: canonical pre-mRNA introns, potentially spliced by the spliceosome; non-canonical pre-mRNA (or mRNA) introns; and short introns within lncRNAs  of the short introns with the most read support are canonical introns–those with GU-AG, GC-AG, or AU-AC sequences at the ends. The majority (319/322) contain the major-class splice site sequences, rather than AU-AC, arguing that these introns may be removed by the major-class spliceosome, although it should be noted that the minor spliceosome can remove introns with major-class splice sites 2 of the . We firsSAMD11, CSNK1G2, FBXW5, and ZNF598 loci, all of which contain canonical short introns. The SAMD11 and CSNK1G2 pre-mRNAs have annotated short introns, and these short introns are contained in pre-mRNAs with multiple smaller introns (SAMD11 and CSNK1G2 introns (with 10 nt from the flanking exons); the most energetically-preferred structures are shown in FBXW5 and ZNF598 pre-mRNAs which are alternatively spliced (FBXW5 and 90 nts for ZNF598). For FBXW5, usage of the more proximal 3ˈ splice site would result in a truncated protein. When the FBXW5 and ZNF598 pre-mRNA introns are folded (including both 3ˈ splice sites), both the annotated and unannotated 3ˈ splice sites are predicted to be close in space to the annotated 5ˈ splice site  lack splice site consensus sequences for either the major or minor spliceosome. A few non-canonical introns  are annotated based on EST data, but the remaining 212 non-canonical introns are unannotated . Most ofXBP1s intron is a non-canonical short intron excised by the RtcB/IRE1 complex [XBP1 as well as ATF4, CES1, GYLTL1B, and RTN4 mRNAs as containing short introns identified in our study. Since there are 21 known IRE1 target mRNAs [CES1, GYLTL1B, and RTN4 are all known RIDD targets.The  complex ,24. We q complex . We idenet mRNAs , we haveWe next asked whether our short intron pool was enriched for the IRE1-mediated mRNA cleavage consensus: CTGCAG ,45. In aAmong the non-canonical introns there are potentially other factors involved in their excision. As a final step, we analyzed the short introns for additional conserved sequences using Multiple Em for Motif Elicitation (MEME ). Apart MALAT1 lncRNA, [MALAT1 contains an unusual 3' end that is formed when RNase P cleaves the transcript [MALAT1 overlaps with the second U-rich motif [MALAT1, promoting MALAT1 degradation. However, we observed high levels of the spliced transcript relative to the unspliced transcript calling that model into question  formation. Recently, there has been intense interest in circRNAs which are produced in an unconventional way by the splicing machinery. With circRNA formation, exons are ligated by backsplicing the 3ˈ splice site of an upstream exon to the 5ˈ splice site of a downstream exon. Further studies showed that extensive base-pairing in the “intron” that would be removed after splicing drove circRNA formation –56. A siHAC1/XBP1 mRNAs that are spliced by the SEN complex and IRE1 . In XBP1s intron [These findings are true of a large number of non-canonical introns, but by no means all of them. One other motif was enriched in the intron pool . Outstans intron –59. FurtHere, we expand the inventory of intronic sequences in the human genome to include many additional short introns. Many more RNA-Seq datasets exist, and our methodology can be extended to these additional datasets making further short intron identification probable. Our analysis includes human cell lines, and it will be interesting to identify tissue-specific short intron splicing as a possible means to alter protein expression. Since most short introns are alternatively spliced, how intron excision is regulated in response to cellular signaling events or in specific tissue environments is an outstanding line of future investigation. Our findings are a next step in understanding an underappreciated aspect of gene regulation, altered mRNA profiles due to short intron removal.http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeCshlLongRnaSeq/. Data were originated from the Cold Spring Harbor Lab as paired-end reads on an Illumina GA2x platform [Human cell line RNA-Seq data were downloaded from the UCSC Genome Browser website: platform . For theGenomic alignment was performed using the STAR alignment algorithm  with defShort introns were identified from the SJ.out.tab output files after STAR alignment. These STAR output files contain information on how many uniquely-aligned reads map across predicted exon-exon junctions. Biological replicates were used to calculate a non-parametric Irreproducibility Discovery Rate (npIDR) as described . npIDR vThe next component of analysis was to analyze predicted introns for sequences that were likely to result in high-frequency genomic deletions such as repetitive sequences. BED files were created with coordinates encompassing ten nucleotides of the predicted 5' and 3' flanking exons as well as the predicted intron. Nucleotide sequences were obtained for these BED files using the Galaxy server and the hg38 genomic assembly.For direct repeat analysis, if six nucleotides of the 5' flanking exon matched the final six nucleotides of the intron, or if the six nucleotides of the 3' flanking exon matched the first six nucleotides of the intron, the predicted intron was labeled a direct repeat and removed from subsequent analysis.For inverted repeat analysis, the intron ends were considered. If the six nucleotides at the 5' end of the intron were complementary to the six nucleotides at the 3' end, the intron was labeled as an inverted repeat and removed from subsequent analysis.i.e. if an intron was >75% repetitive sequence, it was removed from subsequent analysis.For simple repeating sequence analysis, the intron sequences were considered. If an intron contained >75% of a single nucleotide (such as 8 A’s in a 10 nucleotide intron), it was removed from subsequent analysis. These criteria were then applied for any combination of di-, tri-, tetra-, and pentanucleotide repeats, In total, 14,425 predicted introns were analyzed, and 4,008 were removed due to these analyses (note that some predicted introns fell into multiple categories). The remaining 10,417 predicted introns were then compared to longer introns.All introns longer than 70 nts were pooled from the STAR sequencing alignments . Predicted intron ends were then compared to reference intron ends in the hg38 genome assembly to determine whether they were annotated or were unannotated. Introns were separated, and for every intron, total unique reads  and sample number (the number of datasets with an intron) were calculated. Similar analysis was done for all short introns, omitting the annotation stage.As above, BED files were constructed for every short intron remaining in the pool. In each case, three BED files were generated, one with coordinates -15 and -5 from the intron 5' end, one with ten nucleotides centered on the middle of the intron, and one with ten nucleotides +5 and +15 from the intron 3' end. Then reads that aligned to each genomic interval were counted for every BAM alignment file  using the Galaxy server.Splicing efficiency was calculated based on the reads aligning to the intron divided by the average of the reads aligning to either the 5' or 3' flanking exon; this value was then subtracted from 100. All negative splicing efficiencies were set to zero. Both average and maximum  splicing efficiencies were calculated for every intron.CSNK1G2, FBXW5, and ZNF598 mRNAs. Those from the A549 cell line were used to amplify the SAMD11 and MALAT1 RNAs. Primer sequences are: SAMD11 (GGAGATGTTCGCCTGGCAGC and CGTGGTTCAGCACCAGCAGG), CSNK1G2 (AGCAGAGCCGCCACGAC and CTGGGAAGTTCTCGCAGAGC), FBXW5 (TGGCAGGATCTGCTTGATGC and GGCAGACAGCAAGCAAGTCC), ZNF598 (TGGCAGGAGATGGGGTGTCG and GAGCTGCTTAAGCACCTGCG), and MALAT1 (GGCCAAGCTAGCATCTTAGC and TCCTGGAAACCAGGAGTGCC). PCR products were visualized on an agarose gel.For RT-PCR experiments, it was necessary to use a combination of human cell lines, the A549 and HepG2 cell lines. These were cultured under standard conditions: for the A549 cell line  and for the HepG2 cell line . Whole cell RNAs were isolated using TRIzol Reagent according to the manufacturer’s instructions (ThermoFisher Scientific). Reverse transcription was performed using random nonamers for priming and M-MuLV reverse transcriptase, according to the manufacturer’s instructions (New England Biolabs). Reverse transcription reactions derived from the HepG2 cell line were used for PCR amplification of the For the data in unafold.rna.albany.edu). In each case, the minimum free energy was taken for subsequent analysis. Introns were ranked according to free energies and binned in groups of 20 introns, with progressively lower free energies in each subsequent bin. Within each bin, both thermal energies and splicing efficiencies were averaged.To calculate folding energies, every short intron sequence (as well as 10 nt from both the 5ˈ and 3ˈ flanking exons) were analyzed using the Mfold algorithm . Five sequences were sought: CTGCAG, CCGCAG, CAGCAG, CTGCCG, and CTGCAA since these are known IRE1 cleavage sites ,45. 861 meme-suite.org) on all non-canonical introns with a minimum motif size of 6 and a maximum motif size of 10. Only one significant motif was enriched in the non-canonical intron sequences.Short non-canonical introns were also subjected to MEME analysis . MEME waS1 TableFor each predicted short intron, the sequences of the intron and flanking exons were evaluated for repetitive DNA elements.(XLSX)Click here for additional data file.S2 TableFor each cell line, splicing efficiency was calculated and is reported separately for every short intron.(XLSX)Click here for additional data file.S3 TableFor the 600 short introns, with the greatest sequencing read support, we analyzed their location within transcripts as well as whether they were annotated or unannotated in existing genomic assemblies.(XLSX)Click here for additional data file.S4 TableThe short non-canonical introns with the motif from  are pres(XLSX)Click here for additional data file.


The novel method presented here is easy and effective and would provide reference for the preparation of other high performance supercapacitor electrodes.A novel NiO/Ni/RGO three-dimensional core-shell architecture consisting of Ni nanoparticles as core, NiO as shell and reduced graphene oxide (RGO) as conductivity layer, has been constructed by redox reactions with hydrothermal method and heat treatment. High density arrayed nickel nanoparticles (20 nm diameter) semi-coated by a 3 nm thick layer of NiO are evenly distributed on the surface of graphene. This elaborate design not only uses abundant NiO surfaces to provide a wealth of active sites, but also bridges electrochemical active NiO shell and graphene by Ni core to construct an interconnected 3D conductive network. Since both electrochemical activity and excellent conductivity are reserved in this Ni/NiO core-shell/graphene layer 3D structure, the as-prepared electrode material exhibits an extremely high specific capacitance (2048.3 F g Supercapacitors, owing to its ultrahigh power density, fast charge-discharge property and long cycle life, arouse great interest6. Generally, supercapacitors can be classified to electric double layer capacitors (EDLCs) and pseudocapacitors based on different charge storage mechanism. EDLCs exhibit high power density and long cycle life, but low specific capacitance. In contrast, pseudocapacitors show higher specific capacitance and better energy density, but the low utilization rate of active materials and poor conductivity limited its application9. In recent years, transition metal oxides by virtue of high theoretical specific capacitance and electrochemical stability have been studied as electrode materials, such as NiO, MoO3, MnO2, RuO213. Therein, NiO possess high theoretical capacitance (2584 F g−1), low cost and excellent chemical/thermal stability and are considered to be a promising electrode material15. Many efforts have been done to prepare NiO based supercapacitors17, but there is still a certain gap with its theoretical capacitance. Initially, the diffusion distance of electrolytes into NiO electrodes is limited, thus for the bulk structure lacking the diffusion channel, only the surface portion participates in the electrochemical reaction, leading to a less than optimal performance. Furthermore, the poor electrical conductivity of NiO would restrict the fast exchange of electrons. Additionally, any attempt to increase the mass loading usually results in an increase of material thickness, which consequently leads to more active materials being buried and increases the length of active materials away from conductivity base, and correspondingly lower overall efficiencies.With the excessive consumption of traditional energy resources as well as fast-growing market of portable electronics and hybrid electric vehicles, development of high efficiency and sustainable energy storage systems, such as lithium ion batteries, electrochemical capacitors, has been put forward18. For the core-shell structures coated a layer of conductive material  outside the NiO active material, the conductivity does increase significantly, the active material is isolated from the electrolyte. For example, the shell-core structure formed by amorphous carbon coated mesoporous NiO through a hydrothermal method exhibited a specific capacitance of 931 F g−1 at 2 A g−1 19. So, for an expected NiO based core-shell structure as electrode material, increasing the electrochemical active surface while enhancing the conductivity are the key points to improve its electrochemical performance. But so far, these two necessary advantages haven’t been well coordinated.Hence, the thinner thickness of NiO layer, the higher utilization rate of NiO active material. So the NiO based core-shell structures have been widely prepared for supercapacitors, in order to efficiently utilize their rich surface active sites. But the present two kinds of core-shell structure still have some defects. For the core-shell structures fully covered with NiO active material, although abundant sufficient chemically active sites are presented, the electron exchange between every two neighboring core-shell structures was still blocked by at least two layer low conductivity NiO shellIn the present work, we consider a semi-coated core-shell structure for NiO based materials, the core metal is partially coated by active NiO shell and partially connected to the conductive matrix material, to realize the both the electrochemical activity and electron transfer.20. However, Pt is more expensive and may presents weak contact to the NiO21. Therefore, a cheap and efficient conductive material that can bring atomic bonding with both NiO shell and metal core is a prioritized proposal. Recent years, graphene attracted much attention owing to its outstanding electric conductivity, high specific surface area and good chemical stability24. Various approaches to hybrid graphene with nickel oxide proved that it’s helpful to improve the conductivity of supercapacitors27. However, due to the limitation of the amount and distribution of functional groups on graphene, the problem of uneven and low density distribution of NiO particles on graphene arise in NiO/graphene composites, which makes the NiO/graphene electrode materials lacking of sufficient reactive sites and continuous ion diffusion channels. Therefore, an exquisite design of 3D structure of NiO/metal/ graphene to realize fast transfer among high capacitance NiO unites and improve conductivity is critical for ideal NiO based supercapacitor materials.As excellent electric conductive matrix material, Pt has been used as the electron collector to connected the NiO core-shell structure, which exhibits both high energy and power densitiesin-situ oxide in Ni nanoparticles possess excellent structural stability, successfully inhibited the copolymerization between nanoparticles. (2) The connection between Ni cores and graphene facilitate rapid export of electrons, and constructs an interconnected 3D conductive network, using metal Ni cores to bridge electrochemical active NiO shell and conductive graphene sheets. (3) The high density Ni/NiO core-shell nanospheres distributed on graphene provide plenty of electrochemical active sites. (4) The porosity among local high density areas (LHDAs) and the small pores among the nickel particles can create different sizes of channels to benefit the OH− ions diffusion and the occurrence of redox reaction. (5) This design is cost-effective. Such a 3D NiO/Ni/RGO core-shell composite was achieved by a simple hydrothermal and heat treatment method. The composites were characterized and their electrochemical performances were measured to estimate their potential ability as the electrode materials for supercapacitors.Herein, we designed a novel NiO/Ni/RGO three-dimensional semi-coated core-shell architecture for electrode materials of supercapacitors, with high density Ni/NiO core-shell nanospheres dispersing on graphene sheets homogeneously. In this structure, the nickel nanospheres were not completely covered by the shell of nickel oxide due to the parts connecting to graphene sheet was protected by the intense reduction of carbon. Such a deliberately designed structure has several advantages. (1) NiO 16, which signifies that all Ni had been oxide to NiO at such high temperature. While treated at 400 °C (NiONiG-400-2), as revealed by the enlarged picture shown in Fig. Based on the thermo gravimetric (TG) test in Figure 6. The presence of two NiO (NiO-1 and NiO-2) is due to the length of Ni-O bond would changed in the oxidation process. In fact, this is the same kind of NiO. Figure 28. The small binding energy peak located at 535.7 eV is corresponding to C-O. The existence of Ni-O and C-O bonds may mean that both Ni and graphene oxide respectively at the some environment, which may benefit the closely integrated of them. Figure 29. C=C and O-C=O can be regarded as the characteristic functional group of reduced graphene oxide, which proved that the graphene structure was well maintained after 400 °C heat treatment. In addition, no other C-O and C=O related to graphene oxide can be fitted, verifying that graphene oxide is well reduced and consistent with the results of Raman tests in Figure X-ray photoelectron spectroscopy (XPS) spectra of the as-synthesized NiONiG-400-2 composite were shown in Fig. − ions and promote the redox reactions30. Figure 31, which are good in accordance with the XRD result. And the continuous rings without large light spot indicate the homogeneous size of these nanoparticles.The microstructures of RGO/Ni (S-untreated) and NiONiG-400-2 composite were investigated with transmission electron microscopy (TEM). Figure − ions diffusion and improve the electrochemical performance30. After tested by N2 adsorption-desorption isotherms  indicate the revisable redox reaction occurred as followed32:The electrochemical performances of the obtained composites used as work electrodes in a three electrode test system are shown in Fig. −1). The retention of the shape in different scan rates indicates good rate capability of the electrode material, while the shift of redox peaks is attributed to the polarization34. A further examination of rate capability was carried out at a serious of current densities  shown in the Fig. I the discharge is current, Δt is the discharge time, m is the mass of the electrode, and ΔV is the voltage window, respectively. Figure −1, respectively, corresponding to current densities of 1, 2, 5, 10 And 20 A g−1. Chundong W et al.9 by hydrothermal means prepared hierarchical NiO-3D graphene composite delivers a high specific capacitance of 1829 F g−1 at a current density of 3 A g−1, which fully illustrates that NiO/graphene composites have sufficient potential in the preparation of high performance supercapacitor. Xinyi H et al.35 prepared core/shell structure of NiCo2O4@NiCoAl-LDH onto nickel foam and displays a specific capacitance of 1814.24 F g−1 at a current density of 1 A g−1. These performances have a significant advantage over other structures, thus demonstrating that the core-shell structure has its own distinct superiority in the preparation of high performance capacitors. As compared with previous studied NiO based supercapacitors shown in Table −1 reaches to about 80% of the theoretical value of capacitance (2584 F g−1) of NiO.The rate capability of NiONiG-400-2 electrode material was evaluated by CV and galvanostatic charge–discharge tests. Figure s) and the semicircle in the high frequency region reflects the interface charge transfer resistance (Rct) between the electrode and electrolyte, and W, Cdl, Cf are the Warburg impedance, double layer capacitance, and limit capacitance, respectively. Based on the enlarged view of high frequency region in Fig. s and R ct, which indicates that the NiO/Ni/RGO core-shell structure have excellent electron transfer ability and can provides lots of diffusion channels for ions, these are necessary advantages to ensure the ultrahigh specific capacitance of the NiO/Ni/RGO composite.The electrochemical impedance spectra (EIS) measurements for the composites with different treat temperatures and times were carried out from100 KHz to 0.01 Hz with alternate current amplitude of 5 mV in 1 M KOH electrolyte and the results are shown in Figs et al.16 developed a novel ASC (asymmetric supercapacitors) based on carbon nanotube @ nickel oxide nanosheets (CNT @ NiO) core-shell composites for supercapacitor with 93% capacitance retention after 10000 cycles, which shows the excellent stability of the nickel oxide material as the electrode material. The cycling performance of NiONiG-400-2 was tested in the three electrode system at current density of 50 A g−1, shown in Fig. Cycling stability is an important index value to evaluate the electrochemical performance of supercapacitors. Yi, H −1 at current densities of 1 A g−1, about 80% of the theoretical capacitance of NiO (2584 F g−1) and the highest specific capacitance value ever obtained for NiO based materials. This composite also maintains good cycle stability (77.8% capacitance retention after 10000 cycles). From a methodological point of view, the novel method may provide reference for the preparation of other high performance supercapacitor.A NiO/Ni/RGO composite applying for electrode materials of supercapacitors was fabricated by a simple hydrothermal plus heat treatment method. The composite has a 3D structure consisting of Ni/NiO core-shell nanoparticles evenly distributed on the graphene sheets. This unique NiO/Ni/RGO 3D core-shell architecture, using metal Ni cores to bridge electrochemical active NiO shell and conductive graphene sheets, forms a continuous 3D conductive network in the structure and provides a continuous channel for ion diffusion through the gaps and pores among the Ni/NiO core-shell nanoparticles. Therefore, the electrochemical tests demonstrate that the prepared composite possess a very high specific capacitance of 2048.3 F g−1 graphene oxide aqueous solution, a sodium hydroxide solution of Ph = 13 and a 40 mg mL−1 chloroacetic acid aqueous solution were prepared, respectively. The graphene oxide aqueous solution was ultrasound stirred for two hours, then slowly dropwise the sodium hydroxide solution into graphene oxide aqueous solution with volume proportion of 5: 6, stir for 30 minutes for homogeneous mixture. The pH of the mixture solution was adjusted to be 11 with the above mentioned chloroacetic acid aqueous solution, ultra sounding for one hour and stirring overnight. Then, the as-prepared solution was dialyzed until pH was 7. Add 120 mg Polyvinylpyrrolidone into 30 mL as-prepared solution to prevent graphene oxide agglomeration with the metal salt solution. Afterword, a certain amount of nickel sulfate (NiSO4: GO = 1: 8) was dissolved in 10 mL deionized water, and added it slowly dropwise into the above-mentioned solution, stirring for 1 hour. Then, add sodium borohydride solution (600 mg dissolved in 10 mL deionized water) into above-mentioned as-prepared solution slowly. Finally, as-prepared solution was added into the autoclave for hydrothermal reaction at 180 °C for 12 hours followed by natural cooling to room temperature. The synthesized composites were washed with deionized water and alcohol for several times, and dried in vacuum atmosphere at 60 °C, and was labeled as S-untreated (precursor).RGO/Ni composites were synthesized by redox reactions with further hydrothermal method. Firstly, a 2 mg mLIn order to form a NiO layer on the Ni surface, the precursor was subjected to heat treatment at different temperatures for different times. The above mentioned S-untreated precursor was treated at 400 °C for 2 min at air atmosphere with a 5 °C temperature increase rate. After naturally cooled to room temperature, the as obtained sample was labeled as NiONiG-400-2. For comparison, analogous methods were used to treat the precursors at temperature of 250 and 600 °C for 2 min, and 400 °C for 5 and 10 min, respectively. The corresponding samples were labeled as NiG-250-2, NiOG-600-2, NiONiG-400-5 and NiONiG-400-10. The schematic diagram of preparation process is shown in diagram Fig. α radiation (λ = 1.54156 Å), recorded with the 2θ ranging from 20 to 80° at a scanrates of 4°min−1. The thermo gravimetric analysis (TGA) of NiONiG-400-2 was done in NETZSCH STA 449F3. The weight loss of the samples was monitored from room temperature to 700 °C at a heating rate of 10 °C min-1 in nitrogen atmosphere. Raman spectra were obtained on a 514 nm laser beam. The specific surface area was calculated by the Brunauer–Emmett–Teller (BET) method, and pore size distribution was obtained from desorption plots by a Barrett–Joyner–Halenda (BJH) analysis. The field emission scanning electron microscope  and transmission electron microscope  were used to observe the microscopic morphology of the as-prepared samples. X-ray photoelectron spectroscopy (XPS) was used to determine the elements and chemical states in the samples, which was realized on an ESCALAB Mk II (Vacuum Generators) spectrometer with unmono-chromatized Al Kα X-rays (240 W).The crystal structures of the composites were revealed by X-ray diffraction (XRD) using a Rigaku D/max 2500PC diffractometer with Cu K−1, galvanostatic charge–discharge with the constant current density ranging from 1 to 50 A g−1 and electrochemical impedance spectroscopy (EIS) with the constant current density of 50 A g−1 for 10000 cycles were measured in sequence.For preparing electrode, the as-prepared composites, acetylene black and polyvinylidene fluoride at a ratio of 80: 15: 5 were manually grinded adequately for 3 hours with appropriate amount of methylpyrrolidone as mixed solvent, then the slurry were smeared on Ni foam uniformly and dried at 80 °C for 12 hours. The electrochemical performances of the NiONiG-400-2 composites as work electrodes were characterized by a PMC-1000 electrochemical workstation in a three-electrode system using calomel electrode as reference electrode and Pt sheet electrode as counter electrode with 1 M KOH aqueous solution as the electrolyte. Cyclic voltammograms (CV) with the potential window from 0 to 0.7 V at different scanrates varying from 10 to 100 mV sSupplementary Information


In many natural systems, the physical structure of the landscape dictates the flow of resources. Despite mounting evidence that communities’ dynamics can be indirectly coupled by reciprocal among ecosystem resource flows, our understanding of how directional resource flows might indirectly link biological communities is limited. We here propose that differences in community structure upstream should lead to different downstream dynamics, even in the absence of dispersal of organisms. We report an experimental test of the effect of upstream community structure on downstream community dynamics in a simplified but highly controlled setting, using protist microcosms. We implemented directional flows of resources, without dispersal, from a standard resource pool into upstream communities of contrasting interaction structure and then to further downstream communities of either one or two trophic levels. Our results demonstrate that different types of species interactions in upstream habitats may lead to different population sizes and levels of biomass in these upstream habitats. This, in turn, leads to varying levels of detritus transfer (dead biomass) to the downstream communities, thus influencing their population densities and trophic interactions in predictable ways. Our results suggest that the structure of species interactions in directionally structured ecosystems can be a key mediator of alterations to downstream habitats. Alterations to upstream habitats can thus cascade down to downstream communities, even without dispersal. Previous work has shown that directionally biased movement of organisms can have significant effects on species coexistence Levine, , metapopThe understanding of directional resource flows is especially relevant for ecosystems or communities in which resource flows are dictated by gravity or dominant wind patterns, such as river ecosystems, mountain slope habitats, or vertically structured plant communities. For example, the “river continuum concept” , the same bacteria mixture and the bacterivorous ciliate Colpidium sp. (protist “Monoculture”), the same bacteria mixture and Colpidium sp. with the bacterivorous Paramecium aurelia (protist in “Competition”), or with the autotroph Euglena gracilis . To test the sensitivity of our results to initial resource concentration and thus the generality of our findings on the effects of upstream community structure on downstream dynamics, we also replicated our experiment with two different initial inflowing resource levels . Thus, protist communities were added at 20% of their carrying capacity and were allowed to grow 24 hr before the first resource flow event, henceforth referred to as diffusion and described below. In upstream communities with predation, we added five individuals of Daphnia pulicaria in each microcosm. For further details on general methods used in our protist microcosm experiments, see Altermatt et al. (Each microcosm consisted of a 250‐ml Schott bottle that was filled to 100 ml. Microcosms were assembled by first adding 75 ml of pre‐autoclaved and filtered (Whatman filters) standard protist medium, and 5 ml of bacteria inoculum. After 24 hr, to allow time for bacteria growth, we added 20 ml of protist culture with each protist species at carrying capacity  into detritus , and diffusion occurred every Tuesday and Friday. At each measurement day, two 0.5 ml aliquots were sampled for each microcosm: one for protist and one for bacteria density analysis. Protist density was measured by using a standardized video recording and analysis procedure  testing the interactive influence of upstream community structure, presence of a second trophic level in downstream community, and continuous time on log‐transformed bacteria density in the downstream communities. In parallel, for Tetrahymena (thus only in the two‐trophic‐level downstream communities), we performed a two‐way LME testing for the interactive effects of upstream community structure and continuous time on log‐transformed densities. In both models, to control for temporal pseudoreplication issues, we added replicates and time as nested random factors.We analyzed effects of directed resource diffusion on downstream population dynamics of bacteria and Because we were also interested in linking changes in downstream communities to changes in upstream communities, as a complementary analysis, we also tested differences in bacteria and protist densities among the different upstream community structure treatments. To this end, we used a two‐way LME testing for the interactive effects of upstream community structure and continuous time on log‐transformed densities. We also added replicates and time as nested random factors.https://github.com/harveye/Directional_flow).For each LME model, we used an AIC‐based simplification procedure, removing terms sequentially, starting with the highest level of interactions. While we fitted models during model selection using maximum likelihood (“ML”) and the “BFGS” optimization method Nash, , the finhttps://github.com/harveye/Directional_flow.All analyses were conducted with R 3.1.2 , which greatly reduced bacteria densities and dampened variation in population densities across treatments regardless of the upstream community structure  are influenced by the structure of interactions in upstream communities  or a Competition . The average bacteria density of 18.1 × 104 ± 3.7 × 104 ind./μl placed this treatment between the highest (Monoculture and Competition) and the lowest  treatments, in terms of density, which matched with the pattern observed for bacteria downstream where Facilitation also represented the average median density  to the downstream communities, thus influencing their population densities and trophic interactions in predictable ways  in the downstream communities greatly reduced prey (bacteria) density  but not prey (here bacteria) biomass because of prey regulation by the consumer  that feeds both on bacteria and protists. In the facilitation treatment, the presence of the autotroph Euglena gracilis brings in new resource through photosynthesis that benefits bacteria and likely increases grazing top‐down pressure via a bottom‐up trophic cascade—a pattern that we indeed observed in our results: decreasing bacteria density through time  generates a local enrichment effect, albeit limited by this species slow growth rate . Our results thus clarify how upstream communities might affect outflowing subsidy quality/quantity and then cascade spatially to downstream communities. This study also emphasized that measuring detrital content should be a particular concern of future study to further elucidating specific mechanisms.In upstream communities, monoculture and competition treatments had the highest levels of bacteria compared to predation and facilitation. Low bacteria density in the predation treatment can be explained by our use of a large generalist predator , and lowest when growing alone. Although this result does not affect our main conclusion that pertains to the matching patterns between upstream and downstream communities as a function of upstream community structure, it is nonetheless a puzzling observation. Although we can only speculate on this, few hypotheses can however explain this counterintuitive result . Because our bacteria community was composed of three species with wide interspecific size variations, selective feeding by the consumer, releasing one bacteria species from competition, thus leading to higher cell density , appear to be most likely explanation.Interestingly in our upstream ecosystems, we observed that bacteria densities were highest when growing with a consumer (Many natural ecosystems are characterized by directionally biased spatial flows of organisms and resources, such as alpine slopes, seashore habitats, estuaries, vertical structure of tree or plant habitats, and river ecosystems, with the latter likely being the most studied. As opposed to many terrestrial systems, strong directional movements along dendritic‐shaped networks dominate spatial processes in rivers (Altermatt, per se might be a key mediator of spatial changes in community dynamics by indirectly linking communities via directional nutrient flows. This has significant, but yet untested implications for landscape management and the restoration of ecosystem services in ecosystems with directionally biased resource flows.Our results suggest that upstream species interaction networks might be a key mediator of alterations to downstream habitats in directionally structured ecosystems. For instance, the impact of large nutrient loads from agricultural source upstream on downstream lakes could potentially be mitigated or amplified depending on the interaction structure of upstream communities. In our study, we showed that a specific upstream community structure has the same qualitative effect on downstream dynamics regardless of initial resource concentration (non‐diluted resource; Figure None declared.EH, IG, CL, EAF, and FA designed the research; EH and IG conducted the research and processed the data with laboratory support from CL and EAF; EH analyzed the data; all authors participated in results interpretation; EH wrote the first draft of the manuscript; EH and FA edited the first draft; All authors significantly contributed to further manuscript revisions. Click here for additional data file.


G/ID ratio calculated from the Raman study increases with the increasing of the degree of the graphitization in the order of rGO from oil palm leaves (rGOOPL) < rGO palm kernel shells (rGOPKS) < rGO commercial graphite (rGOCG) < rGO empty fruit bunches (rGOEFB) with the IG/ID values of 1.06, 1.14, 1.16 and 1.20, respectively. The surface area and pore volume analyses of the as-prepared materials were performed using the Brunauer Emmett Teller-Nitrogen (BET-N2) adsorption-desorption isotherms method. The lower BET surface area of 8 and 15 m2 g−1 observed for rGOCG and rGOOPL, respectively could be due to partial restacking of GO layers and locally-blocked pores. Relatively, this lower BET surface area is inconsequential when compared to rGOPKS and rGOEFB, which have a surface area of 114 and 117 m2 g−1, respectively.Herein, a new approach was proposed to produce reduced graphene oxide (rGO) from graphene oxide (GO) using various oil palm wastes: oil palm leaves (OPL), palm kernel shells (PKS) and empty fruit bunches (EFB). The effect of heating temperature on the formation of graphitic carbon and the yield was examined prior to the GO and rGO synthesis. Carbonization of the starting materials was conducted in a furnace under nitrogen gas for 3 h at temperatures ranging from 400 to 900 °C and a constant heating rate of 10 °C/min. The GO was further synthesized from the as-carbonized materials using the ‘improved synthesis of graphene oxide’ method. Subsequently, the GO was reduced by low-temperature annealing reduction at 300 °C in a furnace under nitrogen gas for 1 h. The I Green technology is generally considered as a strong stimulant towards economic growth and a means for mitigating environmental degradation, especially in developing countries. Malaysia is one of the world’s largest palm oil producers with about 950,000 ha of land  and oil For this reason and coupled with our quest and curiosity to explore new knowledge, we felt it was essential to devise an experimental means of enhancing the utilization and production of profitable materials from those ‘green wastes’ generated by oil palm industries that can match the ‘wastes to wealth’ mantra, which is focused towards ‘zero-waste industry’.Interestingly, this is occurring also at a time when the world is moving towards green technology. The good prospects of natural precursors as a source of carbon are that they are not only renewable, but also inexpensive and have the potential to be the green alternatives for industrial-scale production of carbon nanomaterials, such as graphene, graphene oxide (GO), reduced graphene oxide (rGO), graphene quantum dots (GQDs), carbon nanotubes (CNT), activated carbon (AC), etc. ,5.In general, carbon nano-structured materials, particularly graphene and other graphene-related and derived materials, are characterized by an extraordinary permutation of exceptional properties developed after a wide range of applications predominantly in areas such as electronics, photonics, energy  and enviMany companies have developed a high level of concern in the processing, modification and customization of these materials and thus have posed a strong need for a method that can be used to characterize these materials. Hence, Raman spectroscopy is one technique that has proven to be appropriate for many of the characterization needs with these materials . For thiIn spite of its short history, the popularity of graphene and its derivatives such as GO and rGO is tremendously spreading every day. This owed to its intense strength and raging electrical, optical and mechanical properties ,14. The It is noteworthy that other semiconductor materials have shown similar structural properties to graphene. Some of them are single crystalline semiconductor films derived from approximately 1 mm-thick, single crystalline wafer templates . Unfortu2SO4, NaNO3 and KMnO4 to oxidize graphite. Since then, much interest was directed to oxidized graphite due to its incredible mechanical, thermal and electrical properties [In this present study, graphene oxide and reduced graphene oxide were successfully synthesized. Captivatingly, graphene oxide is generally considered as the most widely-used substance to produce graphene-like material on a large scale . The cheoperties ,24,25. IDespite all of the good prospects of the GO as the most resourceful and processable graphene precursor, however, there are several challenges in the industrial-scale production of this material, especially in the influence of carbon or graphite source. This drawback has hindered rapid commercialization of GO as compared to chemical vapor deposition (CVD)-graphene due to its complexity. For this reason, GO is mostly processed in combination with other materials, rather than as a finished product for a market with an explicit demand, and its application is also narrowed to where defect-free graphene is not essential . DependiMore so, in spite of all of the promising future of GO for different types of applications due to its incredible processability and dispersibility, it has been reported that the existing chemical oxidation procedures to produce GO are limited by some drawbacks, especially in the formation of permanent hole defects on the GO sheet and residual metal ions contaminations . This ha2 bonding networks in graphene oxide, it is also used as an insulating material [In light of the aforementioned properties, the material is now used in many types of applications. The most prominent ones are, but not limited to, sensors , polymermaterial ,35, althmaterial ,29. Thermaterial ,31,32. Tmaterial .4), sodium borohydrate (NaBH4) [In a chemically-mediated reduction, a variety of chemicals has been used to remove the oxygen groups from the GO . Hydrazi (NaBH4) , etc. we (NaBH4) ,39,40,412) species. The stacked structure is exfoliated by extrusion of the CO2, which is produced when the GO is heated between 200 and 1050 °C. It has been observed that the high temperature gas created intense pressure within the stacked layers. For example, Gonzalez et al. observed structural damage to the GO platelets as a result of the CO2 extrusion during the thermal exfoliation process. In addition, a significant mass of the GO is lost to that effect, and topological defects and vacancies were formed in the rGO structure [The other category or means of reducing the GO is through heat treatment. For example, there are numerous thermally-mediated methods to reduce GO in which the oxide functionalities on the GO are stripped from the surface by directly heating the GO in the furnace under certain conditions and atmosphere . This metructure .3·6H2O and HCl. Their method, though sounding promising, was perceived as tedious and time consuming, and the precursors used are not one hundred percent economical.The most commonly-used material for GO synthesis is high pure graphite flake . Few res3 solution [Another group of researchers, Adolfsson et al., reported to have transformed cellulose materials generated from waste paper into carbon nanospheres via the hydrothermal method . The degsolution . In relasolution .Nevertheless, we were compelled to carry out this research because the mining of high purity graphite flakes, which comprise the foremost raw material utilized to synthesize graphene oxide and reduced graphene oxide, are polluting and very costly as compared to the industrial graphite. Secondly, the cost of graphene oxide in the market is approximately $200 per gram , althougThe biomass materials used as precursors in this research work were natural or bio-agricultural waste generated from palm oil plantation industries in , one of the most abundant biomass resources in the country. The palm kernel shell (PKS) and empty fruit bunch (EFB) were collected from Sri Dewin Oil Palm Mill Sdn. Bhd. , while oil palm leaves (OPL) were collected from Taman Pertanian Universiti, Ladang 8, Universiti Putra Malaysia, .All of the samples were thoroughly washed several times with deionize water, dried in an oven at 70 °C and crushed into a powder.The carbonization or analytical pyrolysis of the starting material was conducted in a furnace under an inert atmosphere of nitrogen gas  of the Raman spectra was used to measure the graphitic character of the as-prepared materials. X-ray diffraction analyses of the as-produced materials were conducted using a powder X-ray diffraction  at room temperature. The scanning range of all the samples was 5 to 40° (2θ), with a scanning rate of 4° min−1. The external surface morphology and microstructure of all of the rGOs were examined using a field emission scanning electron microscope (FESEM) using an FEI Nova NanoSEM 230, . This machine has two operating vacuum modes (high vacuum and low vacuum) to deal with different types of samples and can achieve a magnification of over 500,000×.The Raman spectroscopic analysis of all of the prepared samples was conducted using a ‘Wissenschaftliche Instrumente und Technologie’ (WITec) Raman spectrometer, Alpha 300R . The Raman frequency was acquired with a laser excitation wavelength of 532 nm and an integration time of 5.03645 (s). The intensity ratio between the D-band at around 1365 cm−1 over the range of 400 to 4000 cm−1. The samples were mixed with potassium bromide (KBr) pellets to drop off the concentration of the sample and acquire a better spectrum [−1, and the analysis was carried out at room temperature. Moreover, the surface area and pore volume of the samples were characterized by BET and N2 adsorption-desorption isotherm analyses using BELSorp Mini II . Finally, the Thermogravimetric analysis (TGA) and its derivative (DTG) were conducted using a TGA/DSC 1HT model,  to measure the changes in properties of the as-prepared materials as they are heated, and was carried out within a temperature range between 25 and 1000 °C.The surface functional groups of the GOs and different rGOs were investigated using Fourier transform infrared spectroscopy (FTIR) technique using Thermo Nicolet, Nicolet 6700 model, , at a resolution of 4 cmspectrum . Thus, p−1 and 1585 cm−1, respectively. The G-band of the as-prepared GO shifted towards a higher wavenumber, indicating the oxidation of graphite-like material, which results in the formation of sp3 carbon atoms. However, the D-band is relatively broadened owing to the size reduction of the in-plane sp2 domains at the time of oxidation.Raman spectroscopy is one of the most widely-used analytical techniques to characterize the bonding structure of carbon-based materials. The technique usually imposes very little constraint on the substrate size and is generally nondestructive . As one D/IG) to assess the extent of the graphite cluster size [D/IG ratios measured from the Raman spectrum and the graphitic in-plane microcrystallite size, La, obtained from X-ray diffraction was very much taken into consideration in their studies. Concomitantly, a linear correlation between ID/IG and 1/La was observed and reported following a sequence of measurements on microcrystalline graphite samples with varying microcrystalline sizes [D/IG ratio to evaluate La while investigating properties, such as the degree of disorder in amorphous carbon, nanographite crystal size and the domain for glassy carbon films [D/IG value outsized the maximum value observed in the microcrystalline graphite studies. Consequently, the linear relationship between the ID/IG and the inverse of the microcrystallite size 1/La does not occur when the value is greater than 1.1 [D/IG ratio also increases. This also explains the increase in the intensity of D-band for most amorphous films after annealing at a higher temperature [D/IG undergoes two different behaviors. One of these behaviors is seen at a regime of ‘diminutive’ defect density where ID/IG will increase as a higher defect density generates ancillary elastic scattering. On the other hand, at a regime of high defect density, the ID/IG will start to reduce as an increasing defect density result in a more amorphous carbon structure, hence reducing all Raman bands [Apparently, Tuinstra and Koenig were the first to come up with the idea of using the intensity of the D-band to that of the G-band  at various carbonization temperatures, 400, 500, 600, 700, 800 and 900 °C, were virtually similar in comparison to that of palm kernel shell (PKS), but always lower in terms of I samples .D/IG ratio or its reciprocal (IG/ID) acquired with laser excitation in the visible region is usually used to substantiate the degree of graphitization of carbon-based materials [G/ID values , CO and CO2 gas release from the mainly kinetically unstable functional groups. Concurrently, a moderate mass loss was also noticed between 375 and 900 °C and may be due to the elimination of more stable functionalities. By comparison, rGOOPL had the least weight loss; rGOPKS and rGOEFB had nearly similar weight losses; and GOCG had the highest among them.Thermogravimetric analysis (TGA/DTG) of the materials is presented in It can be recalled that the starting materials (precursors) used in this study were typical plant or biomass materials that were generally known to contain significant amount of lignin, hemicelluloses and cellulose in their structures. Hence, decomposition of these materials as they are heated will give a good understanding of the thermal behavior of the as-generated materials. During the thermal decay process, at the first stage (below 200 °C), a slight weight loss of less than 10% of the biomass materials was observed, due to moisture loss (dehydration) and thermal decomposition reactions to form volatiles and organic molecules. It was reported that thermal decomposition of hemicellulose occurs at temperatures ranging from 150 to 350 °C . In the 2 adsorption/desorption isotherms analyses . Rationally, the acids  used as the chemical oxidation reagents of the starting materials and combined with the low-temperature annealing reduction of the graphene oxide had played a key role in the surface area and carbon deposition efficiency of the resulting materials.Subsequently, taking into consideration the IWe also noted initially that as the temperature increases during the analytical pyrolysis or carbonization of the starting materials prior to GOs and rGOs synthesis, the G-band became more prominent in the Raman spectra. The results also showed that the carbon yield rate decreased as the carbonization temperatures increased. More so, the 002 planes at about 2θ = 23. 6° from the XRD results also give evidence for the graphitic nature of the rGO samples.


Current therapeutics are only able to slow disease progression, therefore there is a need to explore alternative, novel treatment options. There is increasing evidence that the 3′, 5′ cyclic adenosine monophosphate (cAMP) pathway is an important modulator in the development of fibrosis, with increasing levels of cAMP able to inhibit cellular processes associated with IPF. In this study we investigate the expression of Gs-coupled GPCR. The function of selected agonists at expressed receptors was then tested in a cAMP assay, and for their ability to inhibit fibroblast proliferation and differentiation.Using TaqMan arrays we determined that fibroblasts express a range of G2 (PGE2) receptor 2 and 4, melanocortin-1, β2 adrenoceptor, adenosine 2B, dopamine-1, and adenosine 2A receptors were expressed in HLF. Measuring cAMP accumulation in the presence of selected Gs-coupled receptor ligands as well as an adenylyl cyclase activator and inhibitors of phosphodiesterase showed formoterol, PGE2, treprostinil and forskolin elicited maximal cAMP responses. The agonists that fully inhibited both fibroblast proliferation and differentiation, BAY60–6583 and MRE-269, were partial agonists in the cAMP accumulation assay.Expression analysis of GPCR showed that the prostacyclin, prostaglandin EIn this study we identified a number of ligands that act at a range of GPCR that increase cAMP and inhibit fibroblast proliferation and differentiation, suggesting that they may provide novel targets to develop new IPF treatments. From these results it appears that although the cAMP response is important in driving the anti-fibrotic effects we have observed, the magnitude of the acute cAMP response is not a good predictor of the extent of the inhibitory effect. This highlights the importance of monitoring the kinetics and localisation of intracellular signals, as well as multiple pathways when profiling novel compounds, as population second messenger assays may not always predict phenotypic outcomes.The online version of this article (10.1186/s12931-018-0759-2) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) is an age-related, chronic and progressive fibrotic lung disease, with an annual incidence in the UK of 7.44 per 100,000 people, and a median survival of approximately 3 years from diagnosis . An incrA key therapeutic goal in the treatment of IPF is to reduce tissue fibrosis, which may be achieved by enhancing signals that counteract fibrotic processes, and/or inhibit signals and mediators that promote fibrosis. There are at present two drugs that are now approved for use in the treatment of IPF; nintedanib and pirfenidone. Nintedanib inhibits multiple receptor tyrosine kinases (RTK)  and pirfs-coupled G protein-coupled receptors (GPCR) thus activating adenylyl cyclase (AC) and increasing levels of cAMP, inhibit lung fibroblast migration, proliferation and differentiation. For example, prostaglandin E2 (PGE2) acting at PGE2 receptor 2 (EP2) and 4 (EP4) and iloprost acting at the prostacyclin (IP) receptor, were shown to have an inhibitory effect on proliferation and differentiation of lung fibroblasts, via cAMP accumulation and protein kinase A (PKA) activation [As RTK predominantly signal through the Ras/mitogen-activated protein kinase (MAPK) signalling cascade, it is likely that this pathway is responsible for many of the pro-fibrotic responses observed in IPF . There itivation –16. Additivation . Furthertivation , 19.s-coupled GPCR in order to identify potential new targets to treat IPF. We have explored the ability of Gs-coupled GPCR present in HLF to increase global cAMP using high-throughput compatible, homogenous assay kits. We have further explored how the observed increases in cAMP accumulation translate into an ability to inhibit fibroblast proliferation and differentiation, in order to determine whether any of the selected GPCR would be suitable for exploring further as novel targets to treat IPF.The present study was performed in order to evaluate the anti-proliferative capacity of a range of GFoetal bovine serum (FBS), trypsin-EDTA, Hoechst 33,342, RNAqueous kit, TURBO DNase and High Capacity RNA-to-cDNA kit were purchased from Life Technologies . CGS-21680, BMY-45778, BAY60–6583, iloprost, and NECA were purchased from Tocris . Beraprost was bought from Cayman Chemical Company . PDGF-BB was purchased from R&D Systems . MRE-269 (CAS:475085–57-5) and AGN-205204 (CAS:802906–77-0) were synthesized in house at Novartis . Phospho-p44/42 ERK1/2 (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb primary antibody was purchased from Cell Signalling Technology . CF™647-conjugated Affini-pure goat anti-rabbit IgG and CF™647-conjugated Affini-pure goat anti-mouse IgG were purchased from Biotium, Inc., . Pierce™ LDH Cytotoxicity Assay Kit and CellEvent™ Caspase-3/7 Red Detection Reagent were purchased from ThermoFisher . Staurosporine was purchased from VWR International Ltd. . 96-well white ViewPlates, AlphaScreen cAMP and DELFIA proliferations assay kits were purchased from Perkin Elmer Life Sciences , 96-well black ViewPlates were purchased from Corning , and 384-well black, μ-clear Viewplates were purchased from Greiner bio-one . HitHunter™ cAMP assay kits were purchased from DiscoverX . All other reagents were purchased from Sigma Aldrich .− 1 D-glucose, L-glutamine, pyruvate, FBS (10% v/v), and 25 mM HEPES at 37°C, 5% CO2, in a humidified atmosphere. For experiments cells were harvested using trypsin/EDTA and seeded in their sub-culture medium. Two HLF donors were used in the functional studies: HLF d91019001.2 and HLF 9017402.1, both from healthy/non-smoker Caucasian donors.All studies were performed on normal human lung fibroblasts (C-12361), which were purchased from Promocell . HLF were maintained in DMEM supplemented with 4.5 g LT) set at 0.2 for all samples.Expression of GPCR was determined in HLF using high density, 384-well GPCR TaqMan arrays as previously described . RNA fro80 concentration of PDGF (3 ng/mL) for 10 min, at 37°C, 5% CO2. After stimulation, cells were fixed in 4% paraformaldehyde, washed 3× in PBS (with Ca2+/Mg2+), and incubated with permeabilising blocking buffer (dPBS (with Ca2+/Mg2+), 10% FBS (v/v), 0.1% Triton X-100 (v/v)) for 1 h at 37°C. Wells were then washed 3× in wash buffer , 0.05% (v/v) Tween-20) and incubated with rabbit anti-pERK1/2 antibody , and FITC-conjugated Affini-pure goat anti-rabbit IgG (1:1000 dilution in blocking buffer) for 30 min at 37°C. Fluorescence was then quantified on the InCell2000, using DAPI settings to visualize nuclei (0.1 s exp) and FITC settings for pERK (2 s exp).HLF were seeded overnight at 6000 cells/well in 96-well black clear bottom plates then starved for 24 h in the growth medium devoid of all additives. To assess ERK phosphorylation (pERK), cells were incubated in growth medium devoid of all additives  for 30 min with a range of concentrations of forskolin. After this time, cells were stimulated with an EC80 PDGF response.To quantify levels of pERK fluorescence, a standard Multi-Target Analysis algorithm was used in the INCell Analyzer Workstation (v3.7.1). To account for the inter-assay variation in levels of ERK phosphorylation produced in each experiment, data were normalized to the ECw/v HSA, and 5 μM rolipram. Rolipram was included in these assays to inhibit PDE activity, which resulted in increased maximal responses of cAMP accumulation, without any effect on EC50 values generated (data not shown). Rolipram was excluded when measuring cAMP levels in response to PDE inhibitors. cAMP levels were measured using either HitHunter cAMP assay or AlphaScreen competition assay following manufacturer protocol, and were assessed on either a BMG LABTEK ClarioStar or a Packard EnVision plate reader. cAMP concentrations in each well were determined using a standard curve. To account for the inter-assay variation in levels of cAMP produced in each experiment, data were normalized to the maximal forskolin response.HLF were grown to confluency in 96-well white ViewPlates, before incubation with a range of agonist concentrations, or vehicle, for 2 h at room temperature in HBSS with 5 mM HEPES, 0.1% w/v HSA, for 24 h.Proliferation of HLF was measured by incorporation of bromodeoxyuridine (BrdU). HLF were seeded overnight at 4000 cells/well in 96-well black ViewPlates, before being starved for 24 h in culture medium devoid of FBS. Proliferation was measured after incubation with a range of concentrations of FBS or PDGF, in DMEM supplemented with 0.1% For anti-remodelling assays using BrdU incorporation, cells were incubated for 24 h with a range of concentrations of test compounds in the presence of near maximal concentrations of FBS or PDGF in DMEM supplemented with 0.1% w/v HSA. Proliferation was assessed using the DELFIA BrdU incorporation assay kit following manufacturer protocol. Fluorescence was measured using a Packard EnVision plate reader. To account for the inter-assay variation in levels of proliferation in each experiment, data were normalized to the PDGF or FBS response.To assess the differentiation of fibroblasts to the myofibroblast phenotype, immunofluorescence was used to monitor increases in α smooth muscle actin (αSMA). HLF were seeded overnight at 1000 cells/well in 384-well black clear bottom plates, before being starved for 24 h growth medium devoid of FBS.2 in the presence of a near maximal concentration of TGFβ (0.3 ng/mL), to promote differentiation in in DMEM supplemented with 0.1% w/v HSA. Following stimulation, cells were fixed in 4% paraformaldehyde, washed 3× in PBS (with Ca2+/Mg2+), and incubated with permeabilising blocking buffer (dPBS (with Ca2+/Mg2+), 10% FBS (v/v), 0.1% Triton X-100 (v/v)) for 1 h at 37°C. Wells were then washed 3× in wash buffer , 0.05% (v/v) Tween-20) and incubated with mouse anti-α SMA (1:1000 dilution in permeabilising blocking buffer) for 1 h at room temperature, with gentle shaking. Following 3× wash in wash buffer, wells were incubated with blocking buffer containing Hoechst to stain for nuclear DNA (2 μM), and FITC-conjugated Affini-pure goat anti-rabbit IgG (1:1000 dilution in blocking buffer) for 30 min at 37°C. Fluorescence was then quantified on a widefield ImageXpress Micro microscope, with a Plan Fluor 20X objective, DAPI filter cube , 50 ms exposure time for nuclei, and FITC filter cube , 200 ms exposure time for αSMA.For anti-remodelling assays using FMT, cells were incubated for 48 h with a range of concentrations of test compounds and incubated for 48 h, at 37°C, 5% COTo quantify increases in αSMA fluorescence, a standard Multi-Wavelength Cell Scoring algorithm was used in MetaXpress 5.3 software . To account for the inter-assay variation in levels of αSMA expression produced in each experiment, data were normalized to the response to TGFβ.Data were analysed using GraphPad Prism (Version 7.00) and results expressed as mean ± standard error of mean (SEM), unless otherwise stated. Concentration-response data were fitted using a four-parameter logistic equation. Expression of GPCR mRNA is reported as 2 TO - ΔCT compared to the mean Cycle Threshold (CT) of the housekeeping genes and each value is the mean ± SD of 4 biological replicates. The Pearson correlation coefficient (r) was determined using the standard correlation function in GraphPad prism, followed by a two-tailed T test to determine significance.We first aimed to determine whether cAMP accumulation leads to inhibition of the pERK signal cascade in HLF, which may in turn suggest a role for cAMP in the inhibition of proliferation. Inhibition of ERK phosphorylation by the MEK inhibitor PD0325901 blocked PDGF-mediated proliferation of HLF. In turn forskolin, which increases cAMP through direct activation of AC, inhibited PDGF-mediated ERK phosphorylation , β2 adrenoceptor, adenosine 2B (A2B), EP4, dopamine-1, and adenosine 2A (A2A)  that relates to near maximal proliferation  .IPF is driven by a number of pro-fibrotic processes, therefore we next aimed to determine if the same agonists were also capable of inhibiting another key component of fibrosis, TGFβ-induced FMT. TGFβ is a key driver of fibrosis and was able to robustly promote FMT in HLF with ECIn order to test the ability of compounds to inhibit FMT, HLF were treated for 48 h with a range of concentrations of the selected ligands in the presence of a TGFβ concentration (0.3 ng/mL) that relates to near maximal FMT. Agonists and PDE inhibitors inhibited TGFβ-driven FMT with varying levels of efficacy and potency Fig. , however2 = 0.06, p = 0.41), or TGFβ-mediated FMT . This was most apparent with formoterol and treprostinil, which despite generating high levels of cAMP were relatively ineffective at inhibiting proliferation and differentiation. In contrast, BAY60–6583 and MRE-269 exhibited higher efficacy for the inhibition of proliferation and FMT than cAMP accumulation.Because the assays like the simple, homogenous second messenger system used here are often utilised in high throughput screening to identify novel agonists for cAMP accumulation, we wanted to explore the relevance of this method for predicting the inhibition of HLF proliferation and differentiation. To do this, we generated correlation plots for maximal levels of cAMP generation against the maximal inhibition of proliferation or FMT Fig. . There wIPF is a devastating disease, and with current therapeutics only able to slow disease progression and being associated with severe side effects, further research in IPF aetiology and drug discovery is urgently required. A key feature of IPF is airway remodelling which is driven by fibroblast proliferation and differentiation. This leads to the formation of fibroblast and myofibroblast foci that excrete excessive amounts of extracellular matrix resulting in airway stiffening and loss of pulmonary function. It is this airway remodelling that is associated with disease severity , 22 suggs-coupled GPCR in HLF, as well as their ability to increase cAMP and subsequently inhibit HLF proliferation and differentiation in order to identify new targets to treat IPF that will complement existing therapies.Approved therapies and novel approaches undergoing clinical trials in IPF take the approach of inhibiting a range of pro-fibrotic stimuli, often GF-RTK combinations that predominantly signal via the MAPK pathway. Using fibroblast proliferation to model aspects of lung fibrosis, we demonstrated that inhibition of the MAPK pathway completed blocked PDGF-mediated proliferation, and that ERK phosphorylation could be completely inhibited by increasing global cAMP. For this reason, we wanted to explore the expression of Gs-coupled GPCR in fibroblasts, including related receptor families such as those for prostaglandins  and adenosine (A2A and A2B), as well as the MC1, β2 adrenoceptor and dopamine-1 receptors. Importantly, not all of these receptors resulted in functional cAMP responses, as measured by high throughput, homogenous assay systems. This highlights the need to look not only at mRNA levels, but protein expression and function as well to confirm active receptors are present.We observed mRNA expression of a number of different GTo model chronic aspects of IPF we chose to study HLF proliferation and differentiation, as HLF are key mediators of fibrosis and contain human receptors and signalling proteins at relevant levels. Proliferation was monitored using two complimentary methods, BrdU incorporation into newly synthesized DNA of actively proliferating cells, and direct cell counting. Differentiation was monitored using an FMT assay that measures the increased expression of αSMA that indicates a myofibroblast phenotype. Using the BrdU incorporation assay we were able to quantify proliferation in response to both PDGF and serum (which contains multiple growth factors and proliferative stimuli) at an earlier stage compared to the cell counting method. This is likely due to the different sensitivities of the assays as well as the time course of responses being measured. We also observed that full concentration-response curves for the inhibition of cell counting could not be obtained for all ligands. For this reason, we focused on the BrdU incorporation and FMT assays to explore the responses further.2, EP2, EP4, and A2B receptors, as well as forskolin, an activator of AC, were able to robustly inhibit HLF proliferation and differentiation. Interestingly, some agonists such as MRE-269, AGN205204 and BAY 60–6583 were able to fully inhibit either proliferation or FMT, despite being partial agonists for cAMP accumulation. This demonstrates that many of the agonists we have tested in this study have the potential to inhibit multiple processes that contribute to IPF clinically. It is important to note however that these studies were performed on fibroblasts from healthy donors, and therefore these results should be confirmed in fibroblasts from IPF patients, which may differ in their established phenotype.Using these models, we have demonstrated that selected agonists for the IP, βWe were able to demonstrate using bother fibroblast proliferation and differentiation that cAMP accumulation does not correlate with the degree of inhibition of chronic phenotypic responses. This highlights the importance of developing novel treatments for IPF on the basis of their chronic effects rather than acute cAMP responses. For example, although iloprost has been described as a potential treatment for IPF due to its protective effects against bleomycin-induced pulmonary fibrosis in mice , our dat2 has been shown to inhibit cellular markers of fibrosis [2 signalling in lung fibrosis [2 &/or EP4 agonists are strong candidates for novel IPF treatments. Recent studies have also identified the A2B receptor as an important target in the regulation of both acute and chronic lung disease, with agonists being considered for the treatment of acute lung injury and antagonists being considered for the treatment of chronic lung diseases [2B agonist BAY 60–6583, although not very potent, showed robust inhibition of HLF proliferation.A number of the GPCR we identified here have previously been implicated in the treatment of IPF or other chronic lung diseases. The endogenous EP receptor agonist PGEfibrosis , 16, andfibrosis . This cofibrosis , and thediseases –28. In a2 adrenoceptor agonists are widely used for the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD), providing relief by inducing bronchodilation of airway smooth muscle. We have shown here that the long acting β2 adrenoceptor agonists’ salmeterol and indacaterol may also act as anti-fibrotic agents in IPF through the robust inhibition of HLF proliferation and differentiation.Currently, inhaled βAlthough we did not observe any acute cAMP accumulation with the PDE inhibitor IBMX, we did observe some inhibition of both proliferation and FMT at higher concentrations. In the isolated fibroblasts there may not be sufficient basal cAMP turnover in order for PDE inhibitors to substantially increase levels of cAMP during the shorter cAMP assay, but with the extended incubation for the phenotypic assays (24 or 48 h), IBMX may be able to cause some accumulation of cAMP that is driving this inhibitory effect. In support of this, PDE inhibitor use is being explored for IPF and they have been shown to be effective in the bleomycin model , 19. It The mechanisms behind the apparent disconnect we observed between acute cAMP accumulation and inhibition of chronic phenotypic responses is not yet clear, however there are a number of factors that need to be considered. Firstly, proliferation and differentiation were monitored over a longer time period than the cAMP accumulation assays, so it is possible that the agonists tested were degrading over time leading to loss of response. We have previously demonstrated that for treprostinil and MRE-269 > 90% of the original concentration of agonist remains after 24 h incubation with HLF (unpublished data), suggesting that it is unlikely that ligand degradation is contributing to the effect we have seen.Another aspect of GPCR signalling that may be contributing to the responses we have observed is the ability to signal through multiple G proteins or effector molecules. It has now been well characterized that β-arrestin, once recruited to a GPCR, may initiate a cascade of G protein-independent signalling , the bes2 adrenoceptor agonists, which suggests a role for signalling kinetics in mediating chronic phenotypic events. This has been demonstrated previously for the regulation of gene transcription after GPCR activation, where the kinetics or duration of second messenger responses was shown to drive transcriptional activity [The temporal characteristics of the cAMP response can be controlled directly through the kinetics or duration of the cAMP response, or through time-dependent internalization and desensitization of activated receptors. Agonists such as treprostinil and iloprost which are full agonists in the cAMP accumulation assay may undergo a higher degree of desensitization than the partial agonist MRE-269. This could explain why there is a reversal of intrinsic activity for these ligands in the two assays. However, recent evidence from chronic cAMP studies suggests that regardless of their efficacy, all agonists will produce the same relative degree of functional desensitization . In suppactivity , 32.i-coupled S1P1 receptor [2 adrenoceptor [Compartmentalized cAMP signalling may also contribute to receptor/ligand-dependent downstream effects. Selective activation of specific intracellular pools of cAMP can occur through physical intracellular barriers, such as the A-kinase-anchoring proteins (AKAP) –35, or treceptor  and morenoceptor . Further2, EP2, EP4, and A2B receptors have the potential to inhibit pro-fibrotic processes such as fibroblast proliferation and differentiation, implying a potential for disease modifying effects in IPF. From these results it appears that although the cAMP response is important in driving the anti-fibrotic effects we have observed, there appears to be a threshold of cAMP that is required to inhibit proliferation and differentiation over which any further increases of cAMP do not drive further inhibition. For this reason, the magnitude of acute, global cAMP accumulation does not accurately predict the degree of inhibition of proliferation or differentiation suggesting additional factors, such as the spatio-temporal control of cAMP are important. Further research is required to understand this disconnect, which will aid in the discovery of novel, therapeutically beneficial treatments for IPF.In conclusion, in these studies we have demonstrated that selected agonists at the IP, βAdditional file 1:Additional data describing full GPCR expression analysis in human lung fibroblasts and chemical structures of ligands used in these studies. Further characterization of these ligands in an imaging-based nuclear count assay and cell viability assay is presented. (DOCX 1357 kb)