Salmonella enterica, and other organisms lacking RidA, have diverse growth phenotypes, attributed to the accumulation of 2AA. In S. enterica, 2AA inactivates a number of pyridoxal 5’-phosephate(PLP)-dependent enzymes, some of which have been linked to the growth phenotypes of a ridA mutant. This study used transcriptional differences between S. enterica wild-type and ridA strains to explore the breadth of the cellular consequences that resulted from accumulation of 2AA. Accumulation of endogenously generated 2AA in a ridA mutant resulted in lower expression of genes encoding many flagellar assembly components, which led to a motility defect. qRT-PCR results were consistent with the motility phenotype of a ridA mutant resulting from a defect in FlhD4C2 activity. In total, the results of comparative transcriptomics correctly predicted a 2AA-dependent motility defect and identified additional areas of metabolism impacted by the metabolic stress of 2AA in Salmonella enterica. Further, the data emphasized the value of integrating global approaches with biochemical genetic approaches to understand the complex system of microbial metabolism.Members of the broadly distributed Rid/YER057c/UK114 protein family have imine/enamine deaminase activity, notably on 2-aminoacrylate (2AA). Strains of  A common response to stresses and/or changing environmental conditions is the coordinated regulation of genes that ameliorate the stress and exploit changing nutrient circumstances5. Numerous examples of global responses to environmental stress and nutrient availability that are mediated by transcriptional regulation have been described2. Characterization of these responses has contributed to our understanding of not only metabolic strategies but mechanisms of regulation, effector binding, and gene function. Metabolism can be regulated at many levels, but perturbations in the metabolic network are ultimately expected to, directly or indirectly, impact the transcriptome. Thus, the global expression pattern of a microbe can be used to decipher the environment perceived by the cell and understand conditions both internal and external to the organism8.Microbes inhabit environments that are often dynamic with respect to nutrient availability and the presence of chemical and/or physical stresses. A hallmark of microbes is their ability to respond to changing conditions by altering their metabolic network and/or physical characteristics to survive and thrive under new conditions12. In this scenario, if multiple insults generated similar or overlapping transcriptional profiles, it would indicate the perturbations ultimately affected the same part of metabolism. For instance, in a Bacillus subtilis strain harboring a purA mutation that diminishes flux toward adenine biosynthesis, the cell senses a purine limitation and attempts to overcome it via de-repression of the PurR regulon13. Treatment of Bacillus subtilis with the dihydrofolate reductase  inhibitor, Trimethoprim, causes a disruption of C1 unit transfer in the cell, resulting in a bottleneck in purine biosynthesis at the AICAR transformylase  enzyme. In this case, the cell perceives a purine limitation and attempts to overcome it via de-repression of the PurR regulon14. Thus, the shared features of the transcriptional response to both perturbations indicated that the perceived stress in each case was purine limitation.Metabolic imbalance caused by inhibition of a specific enzyme, for instance by exposure to antimicrobial agents or toxic metabolites, often result in transcriptional changes that are triggered as a consequence of the resultant metabolic imbalanceSalmonella enterica, accumulation of the reactive enamine species 2-aminoacrylate (2AA) perturbs the metabolic network by partially inactivating a number of PLP-dependent enzymes, which results in growth phenotypes19. 2AA is generated as a mechanistic intermediate of multiple PLP-dependent enzymes in central metabolic pathways 21. Thus far, the validated targets of 2AA in vivo are PLP enzymes where the enamine attacks the internal aldimine and results in the covalent modification of the PLP cofactor. Depending on the mechanism of attack, an inactive pyruvate-PLP adduct is formed in the active site, or the enzyme itself forms an irreversible bond with the modified PLP22. In either scenario, the affected enzyme is irreversibly damaged, and flux through the pathway to which it belongs is constrained. A summary of the described RidA paradigm of 2AA stress in S. enterica is provided in Fig. S. enterica and E. coli, have multiple RidA paralogs, but to date no physiological role has been identified for these proteins.In in vivo23. The combination of in vitro and in vivo studies in S. enterica showed that at least the PLP-dependent enzymes serine hydroxymethyltransferase , branched-chain amino acid aminotransferase  and alanine racemase  were targets of free 2AA in vivo and their activity is between 40–80% decreased in strains lacking RidA25. The presence of RidA proteins across all domains of life supports a conserved role for this family in quenching reactive metabolites, including 2AA, to prevent damage to metabolic components.Members of the RidA subfamily of the Rid/YER057c/UK114 protein superfamily, are enamine deaminases that quench 2AA and thus prevent the accumulation of this metabolite in vivo is the result of biochemical genetic studies, primarily in S. enterica. Although powerful, this approach demanded that the damage caused by 2AA generated a detectable growth phenotype. It was therefore likely that the impact of free 2AA on the metabolic network was broader than what has been defined by this approach. Additionally, the impact that 2AA damage of known targets has on the global network configuration remains unexplored. For example, while it is known that the growth defect of an S. enterica ridA mutant grown in minimal medium is caused by 2AA damage to serine hydroxymethyl transferase , it is unclear how the cell perceives the resulting reduction in C1 unit production24. The current study was initiated to generate a global view of metabolic changes that result from the accumulation of 2AA in S. enterica. The premise of this work was that a transcriptional profile reflects expression changes that result, directly or indirectly, from perceived changes in the internal or external environment26.The current understanding of 2AA targets S. enterica ridA mutant and the isogenic wild-type strain were defined after growth on minimal glucose medium to provide a snapshot of the global metabolic state of the cells. The transcriptome data were viewed with two questions in mind; i) are previously characterized metabolic defects caused by elevated 2AA discoverable, and ii) do the data generate new insights about the global impact of 2AA accumulation?The transcriptomes of a ridA mutant and wild-type strains, using a false discovery rate (FDR) less than 0.05  and those that showed less than a two-fold difference in expression were excluded to generate the dataset of 84 genes shown in Fig. ilv) were de-repressed in a ridA mutant, despite the fact that the specific activity of isoleucine transaminase  is more than 50% reduced in the absence of RidA18. These data corroborated the conclusion reached with a single reporter, that lack of RidA did not result in a starvation for isoleucine18. Similarly, no differential expression was simply attributable to the decreased activity of serine hydroxymethyl transferase (GlyA) found in ridA mutants. The transcription of dadAX was increased in the ridA mutant and potentially reflected a consequence of the decreased alanine racemase activity of DadX, caused by 2AA accumulation22, since increased accumulation of L-alanine may contribute to the complex transcriptional regulation of dadAX28.Pair-wise comparison of gene expression identified 413 genes with changes  in transcript abundance between 05 Table 27. Of thCT) method29 , one (thiF) had a statistically significant difference in expression that was less than two-fold  showed no statistically significant change in gene expression between mutant and wild-type. For the six genes, a strong positive correlation between results from the qRT-PCR and global RNA-seq approaches was supported by a Pearson’s correlation coefficient of r2 = 0.96. Of the four genes with a greater than two-fold expression difference, only napF had a statistically significant  discrepancy between the RNA-seq and qRT-PCR data. However, both approaches detected a greater than ten-fold decrease in the napF transcript levels in a ridA mutant compared to wild-type. Finally, although RNA-seq transcriptome analysis detected increased sdaC expression in a ridA strain, the expression difference did not meet the statistical cut off (FDR < 0.05). The qRT-PCR data similarly found this transcript increased in a ridA mutant, but the difference was statistically significant using this technique, suggesting that the DEseq algorithm, used to call differentially expressed genes from the RNAseq dataset, performed conservatively30.Results from the global RNA-seq experiment were independently validated with real-time qRT-PCR. The relative expression level of six select genes was measured using the comparative threshold cycle  were differentially expressed in the two strains. Genes in SPI1 and SPI5 generally had lower expression and those in SPI2 had higher expression in the ridA mutant than wild-type strain. Expression of genes involved in chemotaxis, including cheM, cheA and cheY, was also decreased in a ridA mutant . This level of differential expression can generate a detectable motility defect31, and the swimming motility of the wild-type and ridA mutant strains was tested. As predicted by the transcript levels, the ridA mutant was less motile than the wild-type strain on minimal medium , presumably by allosteric inhibition of IlvA33, that decreased the formation of 2AA25. The latter conclusion was further supported by strains carrying the ilvA219 allele, which encodes an IlvAL447F variant, that is resistant to allosteric inhibition by isoleucine9. When the ilvA219 allele replaced wild-type ilvA, data in Table ridA mutation decreased motility by ~35%. Finally, by increasing the production of 2AA, the motility was further decreased (Table ABcc1), there are two copies of ilvA, the wild-type allele in the chromosome and the ilvA219 allele under the control of an inducible PBAD in the chromosome34. Isogenic ridA and ridA+ strains carrying the ABcc1 construct were generated. In this experiment, without induction of ilvA219, the ridA mutant had 20% less motility than the wild-type strain. When expression of ilvA219 was induced, the ridA mutant had a greater than 40% defect in relative motility. Addition of isoleucine partially restored motility, consistent with the presence of the wild-type IlvA, which is sensitive to allosteric inhibition. Taken together the data support the conclusion that like previously characterized phenotypes, the effect of the ridA mutation on motility is due to the accumulation of 2AA generated by IlvA.Metabolic phenotypes of a S. enterica flagellar apparatus is controlled by a well-characterized regulatory cascade, and expression/activity of the master regulator FlhD4C2 is tightly regulated. Figure ridA mutant had decreased expression of several operons that are directly, or indirectly, activated by the FlhD4C2 master regulator. These genes included napFDAG, flg and fli genes and those in SPI I, as indicated in Figure 36. A simple scenario would suggest the function of the FlhD4C2 activator complex was reduced in a ridA mutant, resulting in the decreased expression of its regulon. However, the expression of flhD and flhC were significantly (FDR < 0.05) higher in the ridA strain . These two results could be reconciled if the ridA mutation generated a post-transcriptional effect that resulted in differential function of FlhD4C2 in the two strains.Biosynthesis of the flhDC translation or FlhD4C2-dependent promoter binding, elimination of the respective regulatory component would nullify the effect of the ridA mutation. Lesions in the small regulatory RNAs omrBA, micA, arcZ, oxyS, as well as rpoS, each of which post-transcriptionally regulate FlhD4C2, were introduced to isogenic ridA strains. The motility of the resulting strains was determined, and the data are in Table arcZ and rpoS caused no detectable defect in motility compared to wild-type and did not impact the effect of the ridA mutation  with a ridA mutation further decreasing the motility by ~30%  increased motility of wild-type S. enterica and the impact of a ridA mutation introduced into this background was consistent with the ~30% decrease repeatedly observed  between a ridA flhDC::cat mutant (DM15818) and flhDC::cat wild-type (DM15817)  changed the relative expression (ridA:wild-type) of the napF, fliI, and cheM genes. Strikingly, expression of fliC was not significantly (p < 0.05) different between the flhDC and flhDC ridA strains. These data supported a model that a single perturbation affecting FlhD4C2 activity, directly or indirectly, could be responsible for the expression pattern of genes involved in flagellar biosynthesis, nitrate reduction, and chemotaxis. It is formally possible that 2AA interacts directly with FlhD4C2 to reduce its activity, but since free 2AA is not known to damage enzymes lacking a PLP cofactor, a more likely model suggests a disruption of the metabolic network to which a post-transcriptional regulator of flhDC responds.To investigate the scope of 17) Fig. . Welch’sflhDC mutation did not alter the influence a ridA mutation had on transcription of genes located within SPI1 (invA), SPI2 (ssaV), and SPI5 (sopB). These data suggested that the accumulation of 2AA, caused by the ridA mutation, affected expression of genes in SPI1, SPI2, and SPI5 by an FlhD4C2-independent mechanism. Exogenous glycine eliminates many growth phenotypes of a ridA mutant because it bypasses the need for serine hydroxymethyl transferase (GlyA), which is a primary target of 2AA in S. enterica24. qRT-PCR compared the expression of relevant loci  between ridA (DM3480) and wild-type (DM9404) strains grown on minimal medium supplemented with 1 mM glycine  of the napF, fliI, cheM, invA, and sopB loci was significantly different  when the strains were grown in the presence vs. absence of glycine in the medium. Further, when considered as an isolated experiment, expression of cheM, invA, ssaV, and sopB was not significantly  different between the ridA and wild-type backgrounds, when the cells were grown in medium with glycine. Taken together, these data suggested that exogenous glycine eliminated the effect of a ridA mutation on the expression of cheM, invA, ssaV, and sopB, but napF, fliI, and fliC expression was still significantly  reduced in the ridA mutant. The latter result was consistent with the finding that glycine did not completely restore motility to the ridA mutant  is compromised,  in strains lacking ridA24. By adding glycine and restoring one-carbon unit abundance in a ridA mutant, the differential expression of these SPI loci was minimized. Further work will be needed to extend this correlation and determine whether the ridA paradigm can provide a model to extend the understanding of the intracellular environment and the factors influencing the response of S. enterica to the intracellular environment of the macrophage.Second, the expression pattern of nap) genes in a ridA mutant. Consistent with the expression pattern, ridA mutants had decreased motility, when compared to the parental strain. As with previously characterized phenotypes of a ridA mutant, the lowered motility was a consequence of 2AA accumulation, although the specific target and resulting mechanism were not clear. The only targets of 2AA characterized thus far are PLP-dependent enzymes, where the reactive 2AA enamine attacks the PLP in an active site to lock the respective enzyme in an inactive state22. There are no PLP-dependent enzymes directly involved in motility, suggesting either a new class of targets for 2AA, or an indirect response to a targeted PLP-dependent enzyme(s). The master activator, FlhD4C2, controls a significant number of the genes that are differentially regulated in a ridA mutant, including the periplasmic nitrate reductase (nap) associated genes. The qRT-PCR data showed that the transcriptional remodeling in a ridA mutant is multifaceted, even when only considering pathogenesis-associated genes  toxicity, specifically hypochlorite42. Significantly, ROS stress acts to repress the flagellar biosynthesis machinery43, making it a formal possibility that the chaperon role of RidA is involved in the motility defect described here. However, the genetic data linking the motility defect to 2AA production appeared to eliminate this possibility.In total, the comparative transcription analyses herein identified a new motility phenotype that results from the accumulation of 2AA in Salmonella and uncovered areas of metabolism that may be targeted by metabolic stress caused by 2AA. Identifying additional enzymes that are affected by 2AA will require targeted genetic and biochemical approaches that are driven by global datasets, like the one described here.This study emphasized the ability of transcriptome analyses to provide global metabolic snapshots that may not result in a detectable growth phenotype. It is likely that some of the differentially expressed genes that failed to meet the arbitrary two-fold threshold will prove to be physiologically relevant. These data provided insights into the endogenous environment resulting from the accumulation of 2AA in Salmonella enterica subsp. enterica serovar Typhimurium LT2 and are listed in Table int−201)46. New gene disruptions constructed during this study were made using the λ-Red recombinase system described by Datsenko and Wanner47 using the primers listed in Table 48 to clone the LT2 ridA gene into plasmid pCV1. The resulting construct was confirmed by sequence. pCV1 is a modified pBAD24 plasmid containing BspQ1 sites, conferring ampicillin resistance, and the cloned gene’s expression is inducible by L-(+)-arabinose49. Gene-specific primers for cloning and qRT-PCR analysis were synthesized by Integrated DNA Technologies, Inc.  and are listed in Table S. enterica LT2 gDNA prior to running the qPCR experiment.Strains and plasmids used in this work were derivatives of 450, trace minerals51, and 11 mM D-glucose as the sole carbon source. L-(+)-arabinose was added to a final concentration of 0.2 g/L when needed. Difco nutrient browth (NB) (8 g/L) containing NaCL (5 g/L) was used as rich medium. Difco BiTek agar (15 g/L) was added for solid medium. Ampicillin was added when required for plasmid maintenance to rich and minimal medium at 150 and 7.5 μg/mL, respectively. Chloramphenicol (20 μg/mL) and Ampicillin (150 μg/mL) were used in rich medium, during strain construction. All chemicals were obtained from Sigma-Aldrich Chemical Company .Minimal medium was no-carbon E medium (NCE) supplemented with 1 mM MgSO650) of 0.6. Each 5 mL sample was quickly centrifuged at 16,000 × g in 1.5 mL Eppendorf tubes, supernatant removed and pellets flash-frozen in liquid nitrogen and kept on dry ice. Total RNA was extracted using the RNAsnapTM method52. Pellets were resuspended in 110 μl boil solution  distilled water). Samples were incubated at 95°C for 7 min before centrifuging at 16,000 × g for 5 min while still hot and 100 μl supernatant was transferred to a new 1.5 mL Eppendorf tube. 300 μl UltraPureTM water and 40 μl sodium acetate (3 M [pH5.2]) was added and mixed into each sample before adding 400 μl ice-cold ethanol (100%) and incubating on ice 15 min. Samples were centrifuged at 16,000 × g for 15 min at 4°C and ethanol was decanted. 400 μl ethanol (70% [vol/vol]) was added and pellets were immediately centrifuged at 12,000 × g for 10 min at 4°C before decanting ethanol and pellets were allowed to dry 20 min at 25°C. DNA/RNA pellets were resuspended in 90 μl UltraPureTM water at 4°C overnight prior to treatment with RNase-free Turbo DNase (Ambion), and being precipitated once more by sodium acetate-ethanol treatment. Resulting RNA was resuspended at 4°C for 3 h and stored at −80°C until use.Relevant strains were grown overnight with shaking at 37°C in quadruplicate (RNA-seq) or triplicate (qRT-PCR) in 2 mL NB. Following incubation, strains we diluted 1:100 into 5 mL fresh minimal medium, containing any additional supplements indicated in the text, and allowed to grow with shaking at 37°C to an optical density at 650 nm  following manufacturer’s recommendations. A cDNA library was created from samples containing < 10% rRNA contamination, as measured by the RNA 6000 pico kit for the Agilent 2100 bioanalyzer, and sequenced by the University of Georgia Genomics Facility (GGF) from these RNA samples. Specifically, one hundred nanograms of the rRNA-free RNA was used to create each sequencing library using the KAPA stranded RNA-seq kit (KAPA Biosystems). The RNA libraries were sequenced on an Illumina NextSeq. 500 (150 cycles) Mid Output Flowcell in the paired-end mode with a read length of 75 bp. Processing and differential expression determination of the sequencing data were performed by the Georgia Advanced Computing Resource Center (GACRC) at the University of Georgia. Adaptor removal and quality trimming  of the raw reads were done using FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/) and Trimmomatic (Usadel). The trimmed reads were mapped to Salmonella enterica subsp. Enterica serovar Typhimurium str. LT2 chromosome, complete genome (RefSeq Accession Number: NC_003197.1) and read counts were generated using the prokaryotic-tailored gene alignment tool EDGE-Pro53. Genes differentially expressed  between the respective wild-type and ridA strains of S. enterica were identified using DEseq54. Briefly, an FDR < 0.05 cutoff controls the expected proportion of falsely discovered differentially expressed genes  to 5% (21 genes) of the identified positive hits meeting this threshold (413 genes). The procedures used by DEseq software to determine the FDR value for each gene is described by Benjamini and Yekutieli27.The total RNA was extracted from four independent replicates of S. enterica strain tested, as described in the text, was extracted. An aliquot of each sample was sent to GGF for quality control analysis and quantification using the RNA 6000 nano kit for the Agilent 2100 bioanalyzer and only samples with an RNA integrity number (RIN) over 5.0 were used55. DNase-treated RNA (600ng) was subjected to first strand cDNA synthesis, using the iScript cDNA synthesis kit (Bio-Rad Laboratories) according to the manufacturer’s protocol. PCRs were performed in an Applied Biosystems 7500 Fast real-time (RT) PCR system. Each reaction was carried out in a total volume of 20 μl on a 96-well optical reaction plate (Applied Biosystems) containing 10 μl FastStart Universal SYBR green Master (ROX) mix (Roche Applied Science), 8 ng cDNA, and two gene-specific primers at a final concentration of 0.5 μM each. The real-time cycling conditions were as follows: 95°C for 20 s, and 40 cycles of 95°C for 3 s and 60°C for 30 s. Melting-curve analysis verified that each reaction mixture contained a single PCR product. The threshold cycle (CT) values of gyrB and rpoB were used as internal controls57. Fold changes were calculated using the comparative threshold cycle (ΔΔCT) method29. Briefly, the fold-change in gene expression was calculated using the formula: ridA/wild-type = 2ΔΔCT, where ΔΔCT = ΔCTridA - ΔCTwild-type and ΔCT = CTtarget gene - CTgyrB)normalization gene (. Gaussian error propagation was used to determine the standard error of the mean (SEM) for ΔΔCT from the ΔCT SEM values, and this value used to calculate the 95% confidence interval for each gene. Welch’s two-tailed t-test was performed on log2 transformed 2ΔΔCT values, using GraphPad Prism 6.0b software. To ensure gyrB was constant under the conditions tested, the relative fold change for the internal control rpoB, whose expression was expected to remain constant across treatments, was calculated as described above. Only those samples with an rpoB fold change between 0.8 and 1.25 were used.The total RNA from three independent replicates of each S. enterica cells were grown to full-density in LB and 1 μl of the culture was inoculated onto the plate and incubated at 30 °C inside of a sealed 6 quart plastic Sterilite container to maintain constant moisture content. Motility halos were measured after 20 h using a ruler with markings every mm; if the halo diameter fell between mm markings, the recorded halo diameter was rounded to the nearest marking. Technical replicates  were prepared from each of four biological replicates and the data were averaged and expressed as mean (mm) ± SD.Motility was monitored on minimal medium motility plates (0.25% agar) using either D-glucose (11 mM) or glycerol (20 mM) as the sole carbon source. The plates were prepared in the morning and allowed to solidify at room temperature un-stacked on the benchtop ~6 h before use in the evening. https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103146. All other datasets generated in the current study are available from the corresponding author on reasonable request.Sequencing data were deposited at the NCBI Gene Expression Omnibus (GSE103146), and can be found at Supplementary informationSupplementary Table 1


Selectivity measurements with nucleotide analogues translate our assay into quantitative terms and facilitate drug discovery efforts. The related EBOV and RSV enzymes are not able to efficiently discriminate against ara-cytidine-5′-triphosphate. We demonstrate that this compound acts like a non-obligate chain-terminator.Here we report on the expression, purification and characterization of recombinant ebola virus RNA-dependent RNA polymerase (EBOV RdRp). Active protein complexes composed of the large L protein and viral protein VP35 were isolated from insect cells and analyzed using a short primer/template substrate that allowed benchmarking against related enzymes. RNA synthesis by multiprotein complexes of EBOV, influenza B, respiratory syncytial virus (RSV) and monomeric enzymes of hepatitis C and Zika (ZIKV) viruses required a 5′-phosporylated primer. The minimum length of the primer varied between two and three nucleotides in this system. The EBOV enzyme utilizes Mg Viral RNA-dependent RNA polymerases (RdRp) are essential for replication of RNA viruses and represent important drug targets. Despite recent progress in the field8, the expression of active recombinant RdRp enzymes of negative-sense RNA viruses remains challenging. Influenza (Flu) and vesicular stomatitis (VSV) viral RdRp complexes have been successfully crystallized7 and RNA synthesis of these protein complexes, including the RSV complex, can be monitored in biochemical assays11. However, methods for the study of EBOV RdRp and its inhibition have yet to be devised. The nucleotide analogue (GS-5734) has been shown to be a potent inhibitor of EBOV replication in cell culture12. GS-5734 also provided post-exposure protection in non-human primates. The authors noted that isolation and expression of EBOV RdRp has been elusive and utilized the homologous RSV RdRp to demonstrate enzyme inhibition with the triphosphate form of this compound. Here we report the expression, purification, and biochemical characterization of an active, recombinant EBOV RdRp complex. Multiprotein complexes derived from negative-sense RNA viruses RSV5 and influenza B (FluB)7, and monomeric polymerases derived from positive-sense RNA viruses hepatitis C virus (HCV) and Zika virus (ZIKV) served as benchmarks14.Negative-sense RNA viruses such as influenza viruses, Measles virus, Mumps virus, respiratory syncytial virus (RSV), and ebola virus (EBOV) are important human pathogens. Unfortunately, effective antiviral treatments are often not available7. The three components of this complex  are expressed from the same promoter to yield a single polyprotein, which is cleaved by the co-expressed tobacco etch virus (TEV) protease at engineered cleavage sites. The identity of purified proteins was confirmed by mass spectrometry analysis (MS). Purification of the trimeric complex is enabled through affinity chromatography .Based on the related nature of the RSV and EBOV RdRp complexes, we monitored RNA synthesis activity using a tested RSV-derived, RNA model primer/template (P/T) substrate8 Fig. . The pri8 Fig. . The tem +7 Fig. . We obta20. Motif C . RNA synthesis is here limited by the sub-micromolar concentration of α32P-GTP. Under these conditions HCV RdRp does not initiate RNA synthesis from a di-nucleotide primer  and 2′-deoxy-cytidine-5′-triphosphate (2′-dCTP) that are modified at this position. The 2′-OH group is in the “up” β-conformation in ara-CTP as opposed to the “down” α-conformation in natural CTP pools, while the 2′-OH group is absent in 2′-dCTP , and the product at position +3 is a 7-mer that shows incorporation of ATP at positions +2 and +3 (lane 2). The reaction with ara-CTP shows a product that migrates between positions +3 and +4 (lane 3). The lack of longer reaction products demonstrates effective chain-termination, which is also the proposed mechanism for the triphosphate form of GS-573412. Longer products, including the full-length product are seen in the control in which ara-CTP was replaced with CTP (lane 4). With regards to the incorporation of ara-CTP, RSV, FluB, and HCV enzymes show the same pattern as the EBOV enzyme  and the corresponding value obtained by Deval et al.8 (0.057 ± 0.009 μM). In addition, the selectivity value for ara-CTP in the context of HCV RdRp (100 fold) compares with the previously reported value (140 fold)31. Therefore, this method represents a stable reference system and provides a tool to compare biochemical properties of different RdRp enzymes. EBOV and RSV polymerase complexes show a preference for Mg2+ ions as catalytic cofactors, FluB and ZIKV enzymes utilize Mn2+, and HCV RdRp can utilize either Mg2+ or Mn2+.We further demonstrated that RdRp complexes from EBOV, RSV, and FluB, as well as the monomeric HCV and ZIKV enzymes accepted the same model substrate that was initially described in the context of studies with RSV RdRp L:P complex31. Unexpectedly, specific base-dependent RNA synthesis patterns could be observed only with a 5′-phosphorylated primer. For each of the five RdRp employed in this study, the use of 5′-non-phosphorylated primers yielded reaction products that migrated much higher than expected. While the underlying mechanism remains elusive, this activity is largely independent of the available nucleotides in the reaction mixture. The use of a 5′-phosphorylated primer has been reported in only a few cases, including studies that involved RSV RdRp and mitochondrial RNA polymerase33. Our data show that short model substrates require a 5′-phosphorylated primer to form a biologically relevant elongation complex, which is important for the development of plate-based high-throughput assays that do not reveal the nature of the reaction products.All five enzymes initiate RNA synthesis from a minimum of a 3-mer RNA primer. HCV RdRp was able to utilize a di-nucleotide primer only with high concentration of the initiating nucleotide, which is well-documented in the literature34. Regardless, the biological relevance of our virus specific observations remains to be investigated; however, it has been reported that RNA viruses are able to incorporate dNTP in their genome. The Dengue virus genome contained on average ~7 DNA residues per genome35. Ara-CTP is the triphosphate form of the approved anti-cancer drug cytarabine. Its selectivity value for HCV RdRp reported here (100) correlates well with the previously reported value (140)31. Similar to our measurements with 2′d-CTP, selectivity for ara-CTP also varied among the five RdRp analysed and the order is different. The related EBOV and RSV enzymes show poor discrimination against ara-CTP, which promotes incorporation of the inhibitor under biologically relevant conditions in the presence of competing CTP pools. Moreover, once incorporated, the inhibitor acts like a non-obligate chain-terminator. The ability to cause chain-termination depends on the interaction between the nucleotide analogue and the specific enzyme. Although ZIKV RdRp is able to incorporate ara-CTP, RNA synthesis is in this case only partially inhibited.Active elongation complexes were also analyzed with respect to nucleotide selectivity. To maintain RNA genome fidelity, viral RdRp must select ribonucleotide triphosphates (NTP) against deoxyribonucleotide triphosphates (dNTP) during RNA synthesis. In fact, the five RdRps are all capable of incorporating dNTPs; albeit with varied efficiencies. FluB RdRp exhibited the lowest dNTP selectivity . While this level of selectivity appears to be very poor, it is likely balanced by much higher intracellular NTP pools when compared to dNTP12. The assays described in this study will help to validate the triphosphate form of GS-5734 as a bona fide inhibitor of EBOV RdRp.In conclusion, the present work provides novel biochemical tools for the study of efficiency and fidelity of RNA synthesis by EBOV RdRp. Comparative studies with related enzymes can potentially deliver a platform for drug discovery and development efforts at a larger scale. The use of the same primer/template system facilitates the discovery of both selective inhibitors and other compounds with a broader spectrum of activities. Moreover, the expression of active EBOV RdRp enables mechanistic studies. Experiments with the homologous RSV RdRp suggest that GS-5734 may act as a chain-terminatorEscherichia coli as previously described36. The pFastBac-1 (Invitrogen) plasmid with the codon-optimized synthetic DNA sequences coding for viral proteins or protein complexes (GenScript) was used as a starting material for viral protein expression in insect cells . The construct for the production of the active site mutant D742A EBOV RdRp (L:VP35) was generated with a mutagenic primer. The codon for the amino-acid substitution was introduced with Phusion High-Fidelity DNA polymerase (ThermoScientific) according to the manufacturer’s recommendations. The sequence was confirmed at Molecular Biology Facility at University of Alberta, Canada. We employed the MultiBac (Geneva Biotech) system according to protocols provided by Drs. Garzoni, Bieniossek and Berger38. Viral proteins and protein complexes were purified using the His- or Strep -MS/MS using an LTQ Orbitrap XL hybrid mass spectrometer (Thermo Scientific). Data processing and analysis was done using Proteome Discoverer 1.4 (Thermo Scientfifc) and Sequest (Thermo Scientific), respectively.All virus and viral protein specific information as well as pertinent details of protein expression, purification and RNA synthesis assays are summarized in Supplementary Table The presence of insect cell proteins precluded reasonable estimation of the protein complex concentration in the partially purified EBOV RdRp samples. The RdRp complexes of FluB and RSV did not show significant presence of insect cell proteins Fig. . TherefoThe reactions conditions were chosen so that the RNA synthesis product formation was linear with respect to time. Thus, a 30-minute time point was chosen to stop all the reactions involving titration of nucleotide substrates. Reaction substrates and products were resolved through denaturing 8 M urea 15 or 20% PAGE, visualized and quantified by phosphorimaging. Incorporated nucleotide product fraction was plotted versus nucleotide substrate concentrations and fitted to Michaelis-Menten equation using Prism software (GrapPad).All data generated or analysed during this study are included in the published article (and its Supplementary Information files). Raw numerical values representing PAGE phosphorimager signal quantification are available from the corresponding author on reasonable request.Supplementary Information


Caretakers/parents/caregivers/guardians play important roles in improving Sexual and Reproductive Health (SRH) of adolescents. Caretaker-adolescent sexual communication suggested to influence young people’s sexual behaviours. Despite this significance, the communication is believed to be low in Unguja due to the increase of risky sexual behaviours among adolescents. This study assessed the pattern of such communication using IMB model as a framework.This is a cross-sectional study targeted caretakers of adolescents aged 15–19. One thousand caretakers of adolescents were interviewed using structured questionnaire. Comparison between male and female caretakers on discussing different SRH topics to both sexes of adolescents was made. The mean-score difference of overall communication was examined using Univariate analysis of variance (ANOVA). Bivariate correlation and simple path analysis via regression was conducted to determine the association of IMB variables in relation to communication practice.p < 0.000). Both male and female caretakers mostly discussed sexual abstinence to female adolescents while to male adolescents, HIV/STIs was mostly discussed by female caretakers and pregnancy by male caretakers. The least discussed topics to both sexes are safer sex and other contraceptives use. The bivariate correlations suggested that IMB constructs were inter-related and associated with communication practice.This study finds 40.7% of caretakers had ever communicated with their adolescents on SRH matters and 9.2% reported to have had communicated in the past 30 days. The weighted topic measure revealed only 26.5% of caretakers communicated with their adolescents. Both caretakers communicated more with their female adolescents. The communication was more common between same sex and between caretakers and their biological adolescents (Caretakers-adolescents communication on SRH in Unguja is low and it is not comprehensive. Caretakers fail to communicate with their adolescents on sensitive issues but do so on less sensitive ones. The pattern of communication found to vary across gender of caretaker and that of adolescent and depends on the nature of relationship between caretaker and adolescent. There is gender differences in selecting SRH topics of discussion. Interventions programmes have to include strategies that enhance caretaker’s information, motivation and skills so as to improve SRH communication between caretakers and adolescent. Risky sexual behaviours acquired during adolescence, such as early sexual initiation, unprotected intercourse, and multiple sexual partners, can place young people at risk of HIV infections and sexually transmitted infections (STIs), teenage pregnancy and abortion complications. In Unguja, it was reported that adolescents like to engage in uncontrolled leisure activities, entertainment, music, alcohol, and sexual intercourse at a young age and with different people . By the Parents play a substantial role in the gender development and sexual socialization of their children . Parent-In Sub-Sahara Africa (SSA), studies on caretaker-adolescent communication on SRH are increasing , howeverTheory-based research is necessary to identify the determinants of SRH communication which can be targeted in intervention. The current study utilized Information-Motivation-Behavioral skills (IMB) model  as a fraThe current study therefore assessed caretaker-adolescent communication on SRH using IMB model as a framework. The identification of communication prevalence and the exploration of a relationship of IMB constructs to communication practice will provide pertinent information for planning appropriate intervention programmes for parents to prevent and reduce risky sexual behaviours among their adolescents.National Population and Housing Census of 2012, there were about 302,293 people aged between 35 and 80 years; among them, 147,470 were male and 154,823 were female [This study was conducted in Unguja-Zanzibar, and it involved all the 3 regions of Unguja and all the 6 districts contained within the regions. The study population were all caretakers of adolescents aged 15–19 years. As for this study a caretaker means a parent, caregiver or guardian of adolescent. This definition was conceived because in Tanzanian context, many are extended families and that it is common adolescent live under the care of his/her parents, aunt, uncle, sister/brother or grandparents. According to the e female . Both maWe conducted a cross-sectional community-based household survey among caretakers of adolescents of 15 to 19 years of age to assess their communication practice on SRH with their adolescents. IMB model was used as a framework to guide the assessment process. A total of 1000 from the sampled individuals participated in the study with 206 (20.6%) men and 794 (79.4%) women. Twenty respondents refused to participate due to their personal reasons thus yielding a response rate of 97%.A three-stage probability sampling technique was used to select the individuals. Simple random sampling was used to select 12 wards and 36 shehias. A shehia is the lowest administrative authority in the hierarchy of regional administration . SystemaData were collected from March to April 2014 using face-to-face interviews with structured questionnaire containing closed-ended questions , preventive aspects (e.g. safer sex), and associated risk aspects (e.g. HIV and pregnancy). Participants were required to mention spontaneously the contents and importance of SRH. (e.g. what topics of SRH a caretaker has to discuss with the adolescent? What is the importance of SRH information to adolescents life?) One point was awarded for a correct-match mentioned item. The maximum score of the information construct is 15 points, (Cronbach’s Alpha coefficient was 0.93).Motivation: This was measured with 11 items on (i)Caretakers perceived risk: Three Likert type items were used to assess the caretaker’s perceived risk of his/her adolescents to get reproductive health problems. The question was: Grade the risk of your adolescent female to get  or of your adolescent male to impregnate a girl or to encourage abortion. The score ranged from 1 = no risk at all to 4 = high risk. The maximum score is 12 points in which a high score indicates more feeling of vulnerability for his/her adolescent to experience reproductive health problems (Cronbach’s Alpha coefficient was 0.81). (ii)Caretaker’s social norms: Three Likert type items were used to assess caretaker’s perceptions of social support from significant others in communicating about SRH matters to adolescents . Example of the question was: My friends and relatives talk to their adolescents about SRH matters. The score ranged from 1 = strongly disagree to 4 = strongly agree. The maximum score is 12 points and a high score indicates less restrictive norms in communicating SRH matters with adolescents. Item number 3 was reversed so that higher score meant less restrictive social norms (Cronbach’s Alpha coefficient was 0.41). (iii)Caretaker’s attitudes: Five Likert type items were used to assess caretaker’s attitude towards communicating with adolescents on SRH matters. Example of the question was; Communicating SRH matters with adolescents will promote promiscuity. The score ranged from 1 = strongly disagree to 4 = strongly agree. The maximum score was 20, and all items were reversed so that higher score means positive attitude (Cronbach’s Alpha coefficient was 0.63). The total motivation score was 44 points.Behavioral skills: Behavioral skills in communicating with adolescents about SRH were measured on: (i)Caretaker’s perceived self efficacy: Four Likert scale type items were used to measure perceived easiness in communicating SRH matters with adolescents. Example of the question was: As a female caretaker, I can describe the action of talking to my male adolescent as: The score ranged from 1 = very hard to 4 = very easy, and the maximum score was 16 points (Cronbach’s Alpha coefficient was 0.71). (ii)Caretaker’s perceived Objective skills. Four Likert scale type items were used to measure perceived effectiveness of communicating SRH matters with adolescents. Example of the question was; I can describe my ability to talk to my adolescent as: The score ranged from 1 = very ineffective to 4 = very effective, and the maximum score was 16 points. The total behavioral score was 32 points (Cronbach’s Alpha 0.78).Global measure of caretakers-adolescents SRH communicationWe used this measure to assess whether caretakers had ever communicated with their adolescents on SRH matters. Two questions on likert scale asked how frequently caretakers had communicated with their adolescents  on SRH issues, and how frequently they had communicated in the past 30 days. The response options was 1 = never to 4 = a lot. For the purpose of this analysis, never and hardly ever were recoded as 0 = never, and sometimes and a lot as 1 = ever in both questions.Overall measure of caretakers-adolescents communication on SRHThe overall communication was measured using a weighted topics measure of family sexual communication scale which was developed by Fisher, (1987) . The scaCaretakers-adolescents communication on SRH was assessed using two measures; global communication measure and the detailed examination of communication of specific sexual topics . These procedures were used to facilitate the objective and subjective assessment of parent–child understanding of sexual communication.χ2). The mean score-sex difference of all IMB constructs was examined using a t-test. The overall communication prevalence was computed and the mean difference was compared between different socio-demographic variables using Univariate analysis of variance (ANOVA). Bivariate correlation between IMB variables and communication practice was calculated, and simple path analysis via regression was conducted to determine the association of IMB variables in relation to communication practice. Pearson correlation and regression coefficient was reported and the cut-off point for level of significance was set at a two-sided, p-value <0.05.Data were entered and analysed using Statistical Package for Social Sciences (SPSS) version 22. Descriptive statistics were used, frequency distribution and cross-tabulation used to summarize socio-demographic data and the SRH topics discussed to either sex of adolescents by male and female caretakers, and the difference between them was examined using Pearson’s Chi-square  men and 795 (79.5%) women were interviewed. The age of the respondents ranged from 35 to 80 years with a mean = 45.6 and standard deviation of 10.8. Comparing to male caretakers, significantly more female caretakers were in the age group 35–39 years, and more (77.5% vs. 71.7%) live with their biological adolescents. Male caretakers significantly differed from female caretakers on marital status and occupation as majority of male 193 (94.1%) were married, and 131 (63.9%) were either farmer or fisherman. There were no other significant differences between men and women on other demographic characteristics (Table The results showed that 407 (40.7%) caretakers had ever communicated with their adolescents on SRH issues and 92 (9.2%) caretakers reported to have had communicated with their adolescents in the past 30 days. Female caretakers, caretakers who stayed with their biological adolescents, those of 50–59 years of age, and those having female adolescents were significantly more likely to have had communicated (ever and in the past 30 days) with their adolescents than their counterparts  of caretakers communicated at least four topics of SRH and thus were termed as they communicate. On the other hand, assessment of SRH communication using the weighted topics measure of family sexual communication scale revealed that the participant’s score ranged from 4 to 25 points (maximum 28 pints) with an average of 10.28 and standard deviation of 4.76.This study finds that female caretakers communicated significantly more with female adolescents  and male caretakers communicated significantly more with male adolescents . Moreover, both male and female caretakers who are staying with their biological adolescents communicated significantly more frequent with many topics of SRH to their adolescents than their counterparts. Female caretakers with higher education level and those of age group of 50 to 59 years were more likely to communicate with their adolescents, however there was no significant difference in communication among male caretakers with different education level and within different age groups Table .Table 4MWhen comparing with female, male caretakers communicated significantly more frequent to female adolescents about pregnancy, safer sex, abortion and homosexuality, however; there was no significant difference in communicating different SRH topics to male adolescents among male and female caretakers. The assessment of SRH topics that are mostly discussed by caretakers revealed that abstaining from sexual activities was the most frequently discussed topic to female adolescents by both male 56 (68.3%) and female 209 (63%) caretakers, and it is the least discussed topic to male adolescents by female caretakers 85 (39.2%). HIV/STIs was the most discussed topic to male adolescents by female 103 (47.5%) and pregnancy was most discussed by male caretakers 29 (59.2%), while safer sex (M = 47.6% vs. F = 38.3%) and contraceptive use (M = 39.0% vs. F = 43.7%) were the least discussed topics to female adolescents by both male and female caretakers , Motivation , and Behavioral skills (perceived self efficacy and perceived objective skills in communicating SRH matters with adolescents) were assessed and their pattern of association to communication practice was reported. The findings of the present study represent one of the only quantitative reports which describes the pattern of SRH communication as described by caretakers of adolescents in Tanzania, as many study of parent-child communication relied on the information as reported by adolescents –17.As pointed out in the result, a low proportion of caretakers in Unguja-Zanzibar communicate with their adolescent about SRH issues. The findings show that only 40.7% of caretakers had ever communicated with their adolescents on SRH issues and a very low percentage of 9.2 caretakers reported to have had communicated in the past 30 days. On the other hand, assessment of communication using weighted-specific topics measure revealed that only 26.5% of caretakers reported to have had communicated more frequently with many topics of SRH with their adolescents. Although both communication measures yielded a low communication prevalence, the topic-specific communication measure is more effective and more preferred compared to global form because the global measures may fail to capture the all aspects of sexual communication and may often result in interpretation bias , while tThe low communication prevalence found in this study is not a surprising finding in the context of Zanzibaris cultural background where traditionally, sexual communication is perceived to be a taboo and likely to encourage premarital sexual activities . This prThe communication pattern varies across gender of caretaker and that of adolescent and also depends on the nature of relationship between caretaker and adolescent –33. The Caretakers found to communicate more with their female adolescents rather than male adolescents. One study in Nigeria explored that young women are generally viewed as very prone to deceptions and likely to make mistakes that could ruin their future, that is why parents believed that discussing matters of SRH with female children was more important than doing so with male children . HoweverCommunication about abstinence from sex appeared as the common topic discussed to female adolescents by both female and male caretakers. On the other hand, HIV/STIs was mostly discussed to male adolescents by female caretakers and pregnancy by male caretakers. The rarely discussed topics to female adolescents by both sexes of caretakers are condom and other contraceptive use while to male adolescents, abstinence is rarely discussed by female caretaker and safer sex by male adolescents. Similar topics reported in many studies as the focus of discussion and the least discussed topics between caretakers and adolescents , 34, 36,Results show that caretakers who communicate SRH matters with their adolescents perceived themselves as having high self efficacy, effective objective skills and having positive attitude. These bivariate correlation findings support previous researches , 38 whicThe indicators of motivation in this study show inverse relation with behavioral skills, which is contrary to the IMB model stipulation. In fact those who have less restrictive social norms and positive attitude have low skills and efficacy and communicate less. Similar observation was found in the study  in whichInformation construct on the other hand has a negligible influence on communication. In fact, we observed a considerable variability in communication behavior despite the high score of knowledge on SRH, and therefore we conclude that SRH knowledge alone is insufficient to influence the communication behaviour. This is not surprising, given previous research showing that information is often insufficient to change the behaviour , 41 OtheThe current study finding are likely to have important theoretical and practical implications for refining parent-child SRH communication interventions. However, these implications are constrained by methodological limitations of the current research. Our study, relied on self-report behavioural instruments in which caretakers were asked retrospectively on their communication practice to their adolescents on mentioned topics of SRH without counterchecking their response on the side of adolescents. The quality of such communication was also not determined. Social desirability and recall bias can create response biases and may have lead to over or under -reporting communication behaviours.In conclusion, our findings suggest that caretakers-adolescents communication on SRH in Unguja is low and when it occurs, it is more prevalent among caretakers having female adolescents, having biological relationship with adolescent, and between same sex. Sexual abstinence is mostly discussed to female adolescents by both sex of caretakers, while pregnancy and HIV/STIs are mostly discussed to male by male and female caretakers respectively. Safer sex and contraceptives use were rarely discussed to both sex of adolescents by both sex of caretakers. From this study findings, it is indicated that some communication takes place on particular reproductive health issues but not others. In particular caretakers fail to communicate with their adolescents children on sensitive issues of sexuality like condom use, homosexuality and abortion, but do so on less sensitive ones such as abstinence and HIV. This implies that communication between caretakers and adolescents is not comprehensive and informative when it comes to preparing their growing children to handle the emerging sexual needs they feel responsibly, so that they can minimize the risks for early pregnancy, STIs and HIV infection. Interventions programmes ought to include strategies that enhance caretaker’s information, motivation, skills and confidence in communicating SRH matters so as to improve caretaker-adolescent communication. The current findings are likely to offer new information regarding the use of the IMB model in designing intervention to improve parent-child communication behaviours.


The aim of this study was to find useful metabolites to predict lymph node (LN) metastasis in patients with papillary thyroid cancer (PTC) through a metabolomics approach and investigate the potential role of metabolites as a novel prognostic marker.1H-NMR) spectroscopy. Spectral data by 1H-NMR spectroscopy were acquired, processed, and analyzed. Patients were grouped in three ways, according to the presence of LN metastasis, central LN metastasis and lateral LN metastasis. Chi-square test and the student t-test were used to analyze categorical variables and continuous variables, respectively. The Mann-Whitney U test was used for univariate analysis of metabolites. Orthogonal projections to latent structure discriminant analysis (OPLS-DA) was used for multivariate analysis to discriminate metabolic differences between the two groups.Fifty-two consecutive patients  were enrolled who underwent total thyroidectomy and central LN dissection with or without lateral LN dissection in Severance Hospital between October 2013 and July 2015. The study specimens were provided by the Severance Hospital Gene Bank, and consisted of PTC from each patient. The specimens were prepared for proton nuclear magnetic resonance (Among 52 patients, 32 had central LN metastasis and 19 had lateral LN metastasis. No clinical or histopathological characteristic was significantly different for all comparisons. On univariate analysis, no metabolite showed significant difference for all comparisons. On multivariate analysis, OPLS-DA did not discriminate the presence and absence of LN metastasis. Lactate was found to be the most promising metabolite.No metabolite could discriminate the presence of LN metastasis. However, lactate was found to be the most promising metabolite for discrimination. Further studies with larger sample sizes are needed to elucidate significant metabolites which can indicate the presence of LN metastasis in patients with PTC. The incidence of thyroid cancer has increased worldwide during the last few decades and it is now the most common endocrine malignancy ,2. PapilSeveral prognostic factors have been discovered including patient age at diagnosis, size and extent of the primary tumor, cervical LN metastasis, and occurrence of distant metastasis ,9. Of thV600E mutation results from a single thymine-to-adenosine transversion which is a high specific marker for PTC  were selected for quantification. The peak amplitudes of the metabolites were measured by fitting a Voigt  line-shape function. Metabolites [mM] were quantified by comparing the integrated TSP signal to the metabolite signal.All 1H-NMR spectroscopic data were analyzed using the Mann-Whitney U test because the results of the Kolmogorov-Smirnov test were statistically significant , which indicated that the data did not follow a normal distribution.Patients were grouped into two groups in three different ways, according to the following factors: the presence or absence of LN metastasis, central LN metastasis, and lateral LN metastasis. Normality was assessed using the Kolmogorov-Smirnov test. Chi-square analysis was used to analyze categorical variables and the student t-test was used to analyze continuous variables. www.R-project.org). The muma package was used to perform OPLS-DA [For multivariate analysis of spectral data, Matlab R2012a , SIMCA-P version-13.0 software , and Excel  programs were used. The spectral data were normalized to the total spectral area. The spectral region between 0.5 and 10 ppm was divided into bins of 0.01 ppm width. The water region from 4.6 ppm to 4.9 ppm was excluded prior to analysis. The binned data were aligned using the icoshift algorithm in Matlab , and wer OPLS-DA .Tissue samples were available from 52 patients during the study period. The median age of the patients was 41.5 years (range 15–74 years). Twelve patients were male and 40 were female. The median tumor size was 23 mm (range 13–40 mm). Among the patients, 32 had central LN metastasis and 19 had lateral LN metastasis. All patients with lateral LN metastasis had central LN metastasis as well, thus the results of comparison in two ways, according to the presence or absence of LN metastasis and central LN metastasis were identical. Therefore the comparisons were analyzed in two ways according to the presence or absence of LN metastasis or the presence or absence of lateral LN metastasis. Four patients had distant metastasis.No clinical or histopathological characteristic was significantly different for all comparisons when the patients were classified into two groups according to the presence or absence of LN metastasis or the presence or absence of lateral LN metastasis. Patient demographics and histopathological characteristics are shown in 1H-NMR spectroscopy quantified 24 metabolites in normal and PTC tissues. On univariate analysis, no metabolite showed significant difference between the two groups classified according to the presence or absence of LN metastasis or the presence or absence of lateral LN metastasis (tastasis .OPLS-DA was performed to separate patients into two groups for each comparison. OPLS-DA score plots did not separate the two groups clearly for all three comparisons. When patients were classified according to the presence or absence of LN metastasis and central LN metastasis, the OPLS-DA score plot exhibited nonseparation between the two groups A in . The corThe aim of this study was to explore metabolic differences in PTC according to the presence or absence of LN metastasis. We found that no metabolite could discriminate the two groups. However, lactate was found to be the most promising metabolite for discrimination.Metabolomics is the analytic study of the metabolome, which differs in cancer cells and represents alteration of metabolic processes, and understanding the metabolome will allow deeper understanding of carcinoma development . Several1H-NMR spectroscopy [Subsequent studies focused on discriminating benign and malignant thyroid neoplasms such as follicular adenoma or goiter nodules ,41. Recetroscopy . In thistroscopy . Increastroscopy .1H-NMR spectroscopy. Although our results failed to discriminate patients with and without LN metastasis, our data suggested the possibility of lactate being the most promising metabolite to predict LN metastasis. Lactate has been previously reported to increase in cancer [To our knowledge, this was the first research to discriminate the presence of metastatic LN in patients with PTC using n cancer . Lactaten cancer . Second,n cancer . The impn cancer ,47. In t1H-NMR spectroscopy is the most optimal diagnostic method for the preoperative diagnosis of thyroid nodules and prediction of prognosis. However, as performing in vivo 1H-NMR spectroscopy can be complicated by various issues such as thyroid movement during respiration or shimming difficulty due to large susceptibility differences between the neck and air in trachea, we decided to perform ex vivo 1H-NMR spectroscopy [There are some limitations to this study. First, it is a retrospective study. Second, we performed ex vivo spectroscopy using surgical specimens. In vivo troscopy . Third, In spite of these limitations, our data suggest that lactate may be used to predict LN metastasis and prognosis. Further studies with larger sample sizes are needed to elucidate significant metabolites which can indicate the presence of LN metastasis in patients with PTC.


To control the growth of microorganisms on its surface, human skin produces and releases certain peptides and proteins, including cathelicidin LL-37 and β-defensins. The ribonuclease A (RNase A) superfamily is an important class in this armament  residues , in the nascent RNA. Once bound to the rut site in a newly transcribed RNA, the ATPase activity of Rho is activated, driving its translocation down the RNA. Rho then catches up with the RNA polymerase; the interaction between the Rho protein and the RNA polymerase complex stimulates the dissociation of the transcriptional complex, which results in the termination of transcription. While rut sites are C-rich, a clear consensus sequence has not been identified  may be the reason that targeting the single-stranded poly(C) region of bacterial RNA is enough for human skin RNases to succeed against bacterial invasion. Once the skin RNase cleaves the rut site in a nascent bacterial RNA, the Rho protein can no longer bind to the RNA, and thus will not interact with the RNA polymerase to properly terminate transcription. The author believes she is the first to present the hypothesis that the targeting specificity of human skin RNases may be due to the widely-distributed unstructured C-rich motifs in bacteria, since no related publications have been found to date.Rho-dependent termination accounts for up to half of all transcription termination events in bacteria. Rho-dependent terminators are located at the 3′ ends of genes (intergenic), within the coding sequences of genes (intragenic), and even in the 5′ leader regions   and the desquamin class of RNases are expressed in human skin, and that human skin RNases as a whole specifically cleave RNA substrates at single-stranded C-rich regions (Probst et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA contains over 150 types of chemical modifications. Although many of these chemical modifications were discovered several decades ago, their functions were not immediately apparent. Discoveries of RNA demethylases, along with advances in mass spectrometry and high-throughput sequencing techniques, have caused research into RNA modifications to progress at an accelerated rate. Post-transcriptional RNA modifications make up an epitranscriptome that extensively regulates gene expression and biological processes. Here, we present an overview of recent advances in the field that are shaping our understanding of chemical modifications, their impact on development and disease, and the dynamic mechanisms through which they regulate gene expression. O-methylation (2′OMe) and N1-methyladenosine (m1A) were discovered in tRNA and ribosomal RNA (rRNA); and 2′OMe, N6-methyladenosine (m6A) and 5-methylcytidine (m5C) were found in mRNA and viral RNA [6A, the most abundant internal modification of eukaryotic mRNA, was shown in early studies to facilitate the processing of pre-mRNA and the transport of mRNA [Over 150 unique chemical modifications of RNA have been found in different organisms. The first of these modifications was discovered in 1951, when ion-exchange analysis of RNA revealed an abundant unknown modification later identified as pseudouridine (Ψ) . Discoveiral RNA . As the  of mRNA .6A on RNA, soon followed by that of alkB homologue 5 (ALKBH5), a second m6A demethylase [6A-specific antibodies were used to profile m6A sites through immunoprecipitation followed by high-throughput sequencing. Thousands of m6A sites were identified in human and mouse cell lines, with enrichment around the stop codon and 3′ UTR [We proposed previously that post-transcriptional RNA modifications could be reversible and may significantly impact the regulation of gene expression . This hyethylase , 13. In d 3′ UTR , 15. The6A is installed by a methyltransferase complex that includes the S-adenosyl methionine (SAM) binding protein methyltransferase-like 3 (METTL3), first identified over two decades ago [6A were discovered through experiments in which METTL3 was inactivated; these studies showed that loss of m6A compromises circadian rhythm, embryonic stem cell fate transition, and naïve pluripotency [6A methyltransferase, METTL16, has been shown to regulate the splicing of the human SAM synthetase MAT2A, promoting its expression through enhanced splicing of a retained intron in SAM-depleted conditions, and thus acting as a regulation loop [6A methyltransferase of the U6 small nuclear RNA.mades ago , 17 , an m6A ‘reader’, acts in the nucleus to influence mRNA splicing [6A to promote mRNA processing and alternative splicing [6A readers YTH domain family 1 (YTHDF1) and YTHDF3 affect the translation of their targets through ribosome loading in HeLa cells [6A reader YTHDC2 also functions in the cytosol, affecting the translation efficiency and mRNA abundance of its targets [6A readers, it is becoming evident that their roles may be complex. m6A in the 5′ UTR could facilitate cap-independent translation initiation through a process involving eIF3 [6A from FTO, allowing selective mRNA translation. It will be important to determine the functional roles of readers under different biological conditions.Importantly, msplicing , whereassplicing , 26. In La cells –29, and La cells , 31. The targets . As reseing eIF3 , 34. The6A appears to influence almost every stage of mRNA metabolism. Three recent studies demonstrated interactions with the translation, transcription, and microprocessor machineries . Without m6A, the YTHDC1 homologue YT521-B is unable to properly splice Sxl, leading to failure of X inactivation and thus improper sex determination. Moreover, depletion of Ime4 affects neuronal function, causing shortened lifespan and irregularities in flight, locomotion, and grooming. m6A has also been shown to regulate the clearance of maternal mRNA during the maternal-to-zygotic transition in zebrafish [6A reader Ythdf2 become developmentally delayed because of impaired decay of m6A-modified maternal RNAs. Because these maternal RNAs are not properly decayed, activation of the zygotic genome is also impaired.We recently proposed that mntiation . Such a osophila , 40. Depebrafish . Zebrafi6A in the differentiation of mouse and human embryonic stem cells [6A on differentiation have been shown in mice. Two separate studies showed that the meiosis-specific protein MEIOC, which is necessary for proper meiotic prophase I during spermatogenesis, interacts with the m6A reader YTHDC2 [Meioc are infertile, lacking germ cells that have reached the pachytene phase of meiotic prophase I. Notably, mice lacking Ythdc2 or Mettl3 display similar phenotypes, demonstrating infertility and defects in germ cells, which reach a terminal zygotene-like stage and undergo apoptosis [6A also affects somatic cell differentiation in mice. Knockout of Mettl3 in mouse T cells caused failure of naïve T cells to proliferate and differentiate; in a lymphopaenic adoptive transfer model, most naïve Mettl3-deficient T cells remained naïve, and no signs of colitis were present [Mettl3 caused upregulation of SOCS family proteins, which inhibited the IL-7-mediated STAT5 activation necessary for T cell expansion. Two studies of FTO have also demonstrated roles for m6A in somatic cell differentiation. FTO expression was shown to increase during myoblast differentiation, and its depletion inhibited differentiation in both mouse primary myoblasts and mouse skeletal muscle [Previous studies have demonstrated roles for mem cells , 22, 42.r YTHDC2 , 44. Micpoptosis , 45. m6A present . The lacl muscle . The deml muscle .6A is a critical factor in cell differentiation. Considering that cancer is driven by the misregulation of cell growth and differentiation, it follows that cancer cells may hijack aberrant methylation to enhance their survival and progression. Several studies have demonstrated roles for demethylation or lack of methylation in promoting cancer progression. In MLL-rearranged acute myeloid leukemia (AML), FTO is highly expressed, promotes oncogene-mediated cell transformation and leukemogenesis, and inhibits all-trans-retinoic acid (ATRA)-induced AML cell differentiation [6A methylation and a decrease in the transcript expression of these hypo-methylated genes. ASB2 and RARA are functionally important targets of FTO in MLL-rearranged AML; their forced expression rescues ATRA-induced differentiation. The oncogenic role of FTO is not limited to AML; another study showed that inhibition of FTO in glioblastoma stem cells (GSCs) suppresses cell growth, self-renewal, and tumorigenesis [6A machinery also impact glioblastoma. Knockdown of METTL3 or METTL14 affects the mRNA expression of genes that are crucial to GSC function, and enhances GSC growth, proliferation, and tumorigenesis. In agreement with these findings that lack of methylation tends to promote cancer progression, Zhang et al. [FOXM1 is greatly increased in GSCs as a result of the demethylation activity of ALKBH5; removal of m6A at the 3′ end of FOXM1 pre-mRNA promotes FOXM1 interaction with HuR, which enhances FOXM1 protein expression. A long non-coding RNA (lncRNA) antisense to FOXM1 facilitates the interaction between ALKBH5 and FOXM1, and depletion of either ALKBH5 or its antisense lncRNA inhibits GSC tumorigenesis. ALKBH5 also promotes a breast cancer phenotype; under hypoxic conditions, ALKBH5 expression increases, thus decreasing levels of m6A and upregulating expression of the pluripotency factor NANOG [As discussed in the previous section, mntiation . At the igenesis . This stg et al.  showed tor NANOG .6A methylation tends to facilitate cancer progression, and that RNA methylation could affect cell growth and proliferation. Other studies, however, indicate that the role of m6A in different cancers may be more complex. In hepatocellular carcinoma (HCC), METTL14 downregulation is associated with tumor metastasis, but METTL3 enhances the invasive ability of HCC cells [6A reader YTHDF1. WTAP, a component of the m6A methyltransferase complex, also promotes leukemogenesis, and its levels are increased in primary AML samples [MKL1 [Together, the studies mentioned above suggest that a decrease in RNA mCC cells . SeveralCC cells . Interes samples . RBM15, es [MKL1 .6A machinery can lead to cancer through unique mechanisms. Interestingly, the oncometabolite D-2-hydroxyglutarate (D2-HG), which could act as a nonspecific inhibitor of the iron- and αKG-dependent dioxygenases FTO and ALKBH5, accumulates in about 20% of AMLs [Considering the complex findings, it is likely that different types of cancers can be derived from unique imbalances or misregulation of mRNA methylation. In AML, increased WTAP and RBM15 expression (or writer proteins themselves) could block differentiation, leading to leukemia, whereas increased eraser expression could cause leukemia via separate pathways. The intricate network of interactions is reminiscent of studies of DNA methylation; just as misregulation of DNMT and TET proteins are both associated with cancer –60, misr of AMLs , and mayRecent advances in high-throughput sequencing and mass spectrometry have revitalized research on post-transcriptional modifications, elucidating functions of both known and newly discovered modifications on mRNA Fig. .Fig. 2ChN1 position of adenosine (m1A) was recently discovered on mRNA; this modification was found to occur on RNA at levels around 10–30% of that of m6A, depending on the cell line or tissue [1A occurs in more structured regions and is enriched near translation initiation sites. The level of m1A responds dynamically to nutrient starvation and heat shock, and the 5′ UTR peaks correlate with translation upregulation. As it is positively charged, the m1A modification may markedly alter RNA structure as well as RNA interactions with proteins or other RNAs. Zhou et al. [1A causes A-U Hoogsteen base pairs in RNA to be strongly disfavored, and that RNA that contains m1A tends to adopt an unpaired anti conformation. m1A was also shown to affect translation; its presence at the first or second codon position, but not at the third codon, blocks translation in both Escherichia coli and wheat germ extract systems [1A is present in early coding regions of transcripts without 5′ UTR introns, which are associated with low translation efficiency and which facilitate noncanonical binding by the exon junction complex [1A in translation and RNA–RNA interactions. The exact functional roles of 5′ UTR m1A sites require further studies, and there are also other m1A sites in mRNA that could play distinct roles. Methods to map low abundance m1A sites in mRNA will be crucial to understanding their biological roles [Methylation of the r tissue , 63. m1Au et al.  demonstr systems . In addi complex . These sal roles .O-methylation and m6A methylation to become m6Am, a modification with an unidentified methyltransferase [6Am was recently profiled at single-nucleotide resolution by crosslinking RNA to m6A antibodies and then identifying mutations or truncations in reverse transcription by high-throughput sequencing [6A in vitro and inside cells. However, this is not consistent with the findings of many previous biochemical and cell-based studies [6A demethylation by FTO has been demonstrated biochemically [6A and m6Am, with greater demethylase activity toward m6A modifications that are located internally on mRNA when ultra-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is used to quantify modification changes in a range of different cell lines. Because FTO can work on multiple substrates, including m6Am, and m6Am methylation occurs on only a fraction of all mRNA [Adenosines at the second base of mRNAs can also undergo both 2′-nsferase , 69. m6Aquencing . It undequencing  revealed studies , 71, 72;all mRNA , it willall mRNA , 49, 72.5C was first identified on RNA more than 40 years ago, and is present in all three domains of life [5C on mRNA may, however, produce false positives due to incomplete deamination of unmodified cytidines. Although several biological functions of m5C have been discovered on tRNA (as discussed in the following section), the biological functions of m5C in mRNA have remained largely elusive. Recently, however, a function of m5C on mRNA was recently discovered by Yang et al. [5C promotes nuclear export because it is specifically recognized by the mRNA export adaptor ALYREF. Notably, the study by Yang et al. [5C sites located 100 nucleotides after translation initiation sites, which were not observed by previous studies. Further studies on the enzymes that interact with m5C may lead to the discovery of additional roles for m5C in mRNA.Cytosine methylations are also prevalent in RNA. m of life . It has  of life , 76. Bisg et al. : m5C prog et al.  found en3-Methylcytosine was recently identified as a modification in mRNA, present at a rate of around 0.004% of cytosines in human cell cultures . It is iPseudouridine, which is generated by isomerization of uridine, is the most abundant RNA modification in total RNA . It was tRNAs contain more modifications than any other RNA species, with each tRNA containing, on average, 14 modifications . Recent 1A58 in tRNAiMet and several other tRNA species. m1A58 increases tRNAiMet stability, and its demethylation by ALKBH1 decreases the rate of protein synthesis. A related demethylase, ALKBH3, removes m6A from tRNA and increases translation efficiency in vitro, though its cellular targets and functions have yet to be identified [Liu et al.  recentlyentified .5C on tRNA can also influence translation, particularly affecting stress responses. Deletion of the tRNA m5C methyltransferase NSUN2 reduces tRNA m5C levels and promotes cleavage of unmethylated tRNAs into fragments, which decrease protein translation rates and induce stress response pathways [Nsun2 in mice leads to an increase in undifferentiated tumor stem cells due to decreased global translation, which increases the self-renewal potential of the tumor-initiating cells [Nsun2 also prevents cells from activating survival pathways when treated with cytotoxic agents, suggesting that the combination of m5C inhibitors and chemotherapeutic agents may effectively treat certain cancers.mpathways . Lack ofng cells . Interes5C also plays an important role in the translation of the mitochondrial tRNA for methionine (mt-tRNAMet). m5C is deposited onto cytosine 34 of mt-tRNAMet by the methyltransferase NSUN3 [5C is oxidized by ALKBH1/ABH1 into 5-formylcytidine, which is necessary for reading the AUA codon during protein synthesis.mse NSUN3 –91. Lack5C72 onto tRNA, recognizes both the sequence and shape of tRNA [5C72. C-to-U deamination of C32 in Trypanosoma brucei tRNAThr also depends on multiple factors [3C by two enzymes, the m3C methyltransferase TRM140 and the deaminase ADAT2/3, is a required step in the deamination process. m3C must then be deaminated to 3-methyluridine (m3U) by the same mechanism, and m3U is then demethylated to become U.Methylation and editing of tRNA may require intricate mechanisms and conditions. NSun6, which installs m of tRNA . Without factors . MethylaThe recent discoveries of the first tRNA demethylases, of their effects on translation and differentiation, and of complex mechanisms of tRNA methylation and editing will undoubtedly inspire investigations to elucidate the functions of tRNA modifications and the biological processes to which they respond.Ribosomal RNA is also marked by abundant modifications; the > 200 modified sites in human rRNAs make up around 2% of rRNA nucleotides. Most modifications on rRNA are Ψ or 2′OMe, although rRNA also contains around ten base modifications . Functio6A. Alarcón et al. [6A, which is installed by METTL3 and promotes recognition and processing into mature microRNA by DGCR8. m6A is also present on the lncRNA XIST, and is necessary for XIST to mediate transcriptional silencing on the X chromosome during female mammalian development [6A is present on human box C/D snoRNA species; it impedes the formation of trans Hoogsteen-sugar A–G base pairs, thus affecting snoRNA structure, and also blocks binding by human 15.5-kDa protein [The processing and functions of other non-coding RNA species have recently been shown to undergo regulation by mn et al.  demonstrelopment . Finally protein .6A provides layers of regulation, offering effects that are dependent on the localization of its writers, readers, and erasers.It is becoming increasingly clear that the epitranscriptome and its modifying enzymes form a complex constellation that holds widely diverse functions. Post-transcriptional RNA modifications allow additional controls of gene expression, serving as powerful mechanisms that eventually affect protein synthesis. In particular, m6A machinery can target multiple substrate mRNAs and non-coding RNAs. As we proposed [6A can mark and cause timely expression and turnover of subsets of transcripts. The cellular and compartmental localizations of the writers, readers, and erasers critically affect their functions. Methylation, together with demethylation of subsets of transcripts in the nucleus, may create a methylation landscape that directs the fate of groups of transcripts as they are processed, exported to the cytoplasm, translated, and degraded. Multiple different readers or their associated proteins may be required to actualize the effects of the methylations fully. Although transcript turnover or decay is an accepted role of mRNA m6A methylation, it should be noted that the Ythdf2 knockout mouse exhibits a less severe phenotype [Mettl3 or Mettl14 , demonstrating that the Ythdf2-dependent pathway mediates a subset of the functions of methylated transcripts. There are other crucial regulatory functions of m6A RNA methylation that remain to be uncovered.To facilitate certain cellular processes, the mproposed , cellulahenotype  comparedAlkbh5 does not lead to embryonic lethality but instead causes defects in spermatogenesis [Fto knockout mice display embryonic lethality. Finally, reader proteins act as effectors in a third layer of regulation, carrying out specific functions upon methylated transcripts.These observations lead us to perceive that methylation occurs at multiple layers. Methyltransferases set the initial methylation landscape in coordination with the transcription machinery. Demethylases could more efficiently tune the methylation landscape of a subset of methylated transcripts, acting as the second layer of regulation. Indeed, demethylases often target only a subset of genes under certain conditions; for example, depletion of ogenesis , and onlThe field of epitranscriptomics still remains vastly unexplored. Future studies will need to focus on the mechanisms that define which transcripts are methylated. Moreover, as methylations are often unevenly distributed along the RNA transcript, identifying the mechanisms underlying the regional specificity of methylation, as well as which individual sites along transcripts are methylated, remain as major challenges. The methylation selectivity on particular transcripts may need to be coupled with transcription regulation. How this selectivity is determined and the interplay between methylation and transcription require further exploration. Questions regarding the effects of methyltransferases and demethylases on nuclear processing, splicing, and export also remain. Nuclear regulation of RNA methylation could play critical roles impacting biological outcomes. In particular, it will be important to determine how and why a subset of RNAs undergoes demethylation inside the nucleus, as well as the functional consequences of this required demethylation on gene expression. Interactions between the writers, readers, and erasers with other cellular components are also necessary to reveal functional roles, especially those in complex biological processes in vivo.


A few reports have described the effectiveness of resection for recurrent cholangiocarcinoma. However, none have described resection of synchronous pulmonary metastasis from distal cholangiocarcinoma. We report the first case, to the best of our knowledge, of a slow-growing synchronous pulmonary metastasis from distal cholangiocarcinoma resected 3.5 years after the initial surgery.A 67-year-old Japanese man with a diagnosis of distal cholangiocarcinoma was referred to our hospital. Thickening of the distal bile duct and an air-space pattern in the upper lobe of the left lung were detected by preoperative computed tomography. He underwent pancreaticoduodenectomy for the distal cholangiocarcinoma. Follow-up chest computed tomography demonstrated that the air-space pattern in the left lung had gradually enlarged. Thoracoscopic left S6 segmentectomy with lymph node dissection was performed 3.5 years after the initial surgery. Histopathology of the resected specimen revealed a solitary metastasis from distal cholangiocarcinoma with lepidic growth. We diagnosed the patient with a solitary synchronous pulmonary metastasis from distal cholangiocarcinoma.Surgical resection might offer better long-term survival to patients with synchronous pulmonary metastasis from distant cholangiocarcinoma than nonsurgical treatments. Pulmonary metastasis from distal cholangiocarcinoma may exhibit a lepidic pattern. Therefore, because of the possibility of synchronous pulmonary metastasis, pulmonary resection should be considered for patients with lepidic lesions who have been diagnosed with distal cholangiocarcinoma. Surgical resection is necessary for curative treatment of distal cholangiocarcinoma. Although systemic chemotherapy is applied to distal cholangiocarcinoma with factors of unresectability such as metastasis to the liver, lung, peritoneum, and distant lymph nodes (LNs), its clinical value remains unsatisfactory , 2. A feA 67-year-old Japanese man who had been diagnosed with distal cholangiocarcinoma at a previous hospital was referred to our hospital for surgical treatment. He was a current smoker. His medical and family histories were unremarkable. He had undergone percutaneous transhepatic biliary drainage at a previous hospital because of obstructive jaundice without biliary tract stones. Abdominal computed tomography (CT) revealed thickening of the distal bile duct Fig. , and cheThe patient underwent pancreaticoduodenectomy (PD) for the distal cholangiocarcinoma. Because of its small size, the lesion in the left lung could not be diagnosed accurately in terms of whether it was malignant and had the possibility of being pneumonia. Therefore, the lesion was observed. Histological examination of the resected specimen revealed well-differentiated adenocarcinoma invading the pancreas with five regional LN metastases Fig. . Immunoh18F-fluorodeoxyglucose CT detected abnormal uptake in the left lung lesion with a maximum standardized uptake value of 4.30 (Fig. Follow-up chest CT was periodically performed about every 4 months during the first 5 years after the initial surgery. Follow-up chest CT demonstrated that the air-space pattern in the upper lobe of the left lung had gradually enlarged to 36.8 mm in diameter with no additional lesions for 3.5 years after PD Fig. . The patWith differential diagnoses of either primary lung cancer or solitary pulmonary metastasis from distal cholangiocarcinoma, thoracoscopic left S6 segmentectomy with LN dissection was performed. Histologically, the resected lung specimen showed adenocarcinoma tissue characterized by the formation of irregular tubular structures with lepidic growth Fig. . ImmunohTo the best of our knowledge, this is the first report of resection of a solitary synchronous pulmonary metastasis with lepidic growth from distal cholangiocarcinoma 3.5 years after PD. This patient’s course suggests two important clinical issues. First, surgical resection of a synchronous pulmonary metastasis from distal cholangiocarcinoma might offer better long-term survival than nonsurgical treatments. Although surgical resection is the only curative treatment for distal cholangiocarcinoma, more than half of patients who undergo curative resection develop recurrences , and theSecond, pulmonary metastasis from distal cholangiocarcinoma may exhibit lepidic growth, making it difficult to distinguish from primary lung cancer. In general, pulmonary metastasis is typically described as one or multiple nodules on radiological imaging. However, metastatic pulmonary adenocarcinoma rarely shows lepidic growth on radiological or histopathological examination , 9. ChesWe report the first case, to the best of our knowledge, of a slow-growing synchronous pulmonary metastasis with lepidic growth from distal cholangiocarcinoma resected 3.5 years after the initial surgery. This case is also extremely rare in that we were able to observe the course of the synchronous pulmonary metastasis for a long period of time. Surgical resection might offer better long-term survival to patients with synchronous pulmonary metastasis from distal cholangiocarcinoma than nonsurgical treatments. Pulmonary metastasis from distal cholangiocarcinoma may exhibit a lepidic pattern, making it difficult to distinguish from primary lung cancer. Therefore, pulmonary resection should be considered for patients with lepidic lesions who have been diagnosed with distal cholangiocarcinoma because of the possibility of synchronous pulmonary metastasis.


Aesthetic value, or beauty, is important to the relationship between humans and natural environments and is, therefore, a fundamental socio-economic attribute of conservation alongside other ecosystem services. However, beauty is difficult to quantify and is not estimated well using traditional approaches to monitoring coral-reef aesthetics. To improve the estimation of ecosystem aesthetic values, we developed and implemented a novel framework used to quantify features of coral-reef aesthetics based on people's perceptions of beauty. Three observer groups with different experience to reef environments  were virtually immersed in Australian's Great Barrier Reef (GBR) using 360° images. Perceptions of beauty and observations were used to assess the importance of eight potential attributes of reef-aesthetic value. Among these, heterogeneity, defined by structural complexity and colour diversity, was positively associated with coral-reef-aesthetic values. There were no group-level differences in the way the observer groups perceived reef aesthetics suggesting that past experiences with coral reefs do not necessarily influence the perception of beauty by the observer. The framework developed here provides a generic tool to help identify indicators of aesthetic value applicable to a wide variety of natural systems. The ability to estimate aesthetic values robustly adds an important dimension to the holistic conservation of the GBR, coral reefs worldwide and other natural ecosystems. This esannually . Howeverannually –10 if itannually .Australia's Great Barrier Reef (GBR) was designated a World Heritage Area in 1981 and is widely recognized for ‘containing superlative natural phenomena or areas of exceptional natural beauty and aesthetic importance’ (Criterion vii) . Under tThe perception of beauty is much broader than visual satisfaction; it also includes the sense of pleasure evoked by experiences related to sight, touch, sound, taste or smell ,13. For Petrogale penicillata) in Australia [Panthera onca) in Peru [Immersion via virtual reality (VR) is the process of replacing real sensory input with inputs from a computer system, such that the person is unaware of their outside reality. Immersive VR environments can be 360° images or synthetic three-dimensional environments, using head mounted displays responding to user movements. Immersion in VR places a person in a situation similar to where their knowledge was developed, thereby activating emotions and knowledge linked to those past experiences . As a reustralia  and jagu in Peru . HoweverAccordingly, we developed a VR platform using 360° imagery from the GBR and used it to elicit information about people's perceptions of reef beauty. We interviewed three groups of observers with different experiences of coral reefs: (i) Marine Scientist, composed of experts in coral-reef ecology with extensive personal experience and scientific knowledge of coral reefs; (ii) Experienced Diver, who have extensive diving experience in coral-reef environments; and (iii) Citizen, who for the most part have only experienced coral reefs through documentaries and images. The goals of the elicitation were to (i) identify quantifiable attributes of reef aesthetics and (ii) determine whether these attributes differed among the groups of observers elicited. A Bayesian hierarchical logistic regression model  was deve2.2.1.A total of 39 360° images collected throughout the GBR in 2012 were provided by the XL Catlin Seaview Survey . The imaWe then developed a random sampling design, stratified across each of the reef clusters  that ens2.2.Based on the reef-aesthetic attributes described above, we prepared eight questions with yes/no responses using non-technical language that could be easily understood by all observer groups . Note th2.3.We used VR to elicit information about reef aesthetics from a total of 37 Marine Scientists, 32 Experienced Divers and 36 Citizens between late September and mid-November 2016. The 105 participants represented a range of ages (18 to over 45), gender  and underwater experiences . Each of the participants was given a training document to read, which described the interview questions . The elicitation sequence always started with the same ‘training image’, which was selected because its aesthetic attributes fell into medium-range categories. The reef habitat presented in the training image was characterized by high visibility, structural complexity and colourfulness. Its coral cover and health were low to medium  , with no2.4.We constructed a hierarchical Bayesian logistic regression model to quantify aesthetic values of these images based on the relationships between aesthetic perception and attributes elicited from the participants . In total, eight aesthetic and five demographic attributes were used as explanatory variables . We then3.3.1.Overall, the participants found most images aesthetically pleasing . At the 3.2.For the reef environments that were unanimously perceived as pleasant or unpleasant, we examined how the three groups of participants scored aesthetic attributes between the three reef clusters . In this3.3.The purpose of the model was to estimate the probability that an observer found a reef aesthetically pleasing  and to quantify the relationship between this response and the explanatory variables representing aesthetic attributes and demographic variables . The hig4.Aesthetic ecosystem services describe the human psychological well-being and satisfaction provided by beautiful ecosystems ,27. Howeand reef health irrespective of who is observing a coral reef.Our results showed that high structural complexity of coral reefs strongly increased their aesthetic value. Structurally complex reefs are also considered healthy because they provide a diverse array of habitats. In addition, the loss of structural complexity has detrimental effects on biodiversity –33. MariCoral-colour diversity was also positively associated with high reef-aesthetic value, which confirms assumptions made in previous reef-aesthetic studies that humans prefer colourful reefs ,34,35. HWe found no evidence supporting our initial hypothesis that a person's past experiences influence their perception of beauty. Although this may be due to the relatively small number of observers (maximum of 37 per group), there was a significant amount of agreement across groups about attributes of reef aesthetics. Additionally, our results were similar to those of Dinsdale & Fenton , who fouThe combined use of VR and modern statistical modelling can be easily applied to elicit information in other domains for conservation purposes including social benefits and educational and environmental outreach opportunities for coral reefs, in addition to other similar remote ecosystems that are difficult or expensive to physically access. For example, the ability to immerse citizens into virtual coral reefs is likely to create new insights regarding coral-reef knowledge compared with two-dimensional images or high-resolution underwater video, which are typically shown in the media. In addition, the VR experience could be used to increase the public's understanding of environmental pressures on coral reefs  and theiThe framework developed here provides exciting new opportunities to study aesthetics as an ecosystem service ,10,29. A