As a result, discussion of controversies or changes in practice within radiation oncology involves input from all three disciplines. For this reason, significant effort has been expended recently to foster collaborative multidisciplinary research in radiation oncology, with substantial demonstrated benefit., 2 In li2High linear energy transfer (LET) radiotherapy has long held the promise of improved efficacy against tumors that are refractory to conventional radiotherapy. While such an increase in efficacy is achievable with fast neutron therapy, poor physical dose distribution characteristics have limited its potential. Carbon ions are an elegant solution in that they combine the biological effectiveness of fast neutron therapy with physical dose shaping characteristics even better than proton therapy. However, the cost of technology to deliver this treatment is tremendous and its clinical potential remains largely unproven. Clinical trials are underway in several countries, helping us gather the necessary data to demonstrate its efficacy. However, the United States, a traditional world leader in the development and implementation of advanced healthcare technology, is not among them. Considering the substantial potential benefit of this treatment and also our current efforts to contain the costs of healthcare, is now the time for the United States to step from the sidelines to participate in this research? This is the subject of this month’s 3DCRT debate.Arguing for the proposition will be Drs. Eleanor Blakely, Bruce Faddegon, and Christopher Tinkle. Dr. Blakely is a senior staff in biophysicist (rehired retiree since 2015) at the Lawrence Berkeley National Laboratory (LBNL) with more than 44 yr of professional experience in molecular, cellular, and animal radiobiological research directed at studying the basic mechanisms of radiation responses, with an emphasis on charged particle radiation effects. Dr. Faddegon is a professor of medical physics in the radiotherapy department of UCSF. His research focus is to bring technical innovation into the clinic to improve radiotherapy by advancing linear accelerator, imaging, and particle therapy equipment and methods including Monte Carlo simulation tools and techniques. Dr. Tinkle is a radiation oncologist and Assistant Member in the department of radiation oncology at St. Jude Children’s Research Hospital. His focus is on preclinical and translational studies in pediatric cancers exploring the interactions of proton and photon radiotherapy and emerging targeted systemic therapy.Arguing against the proposition will be Drs. Charles Bloch, Michael Dominello, and Robert Griffin. Dr. Bloch is a medical physicist who started his career in proton therapy over 25 yr ago. Currently, he is an associate professor in the department of radiation oncology at the University of Washington and serves as an associate director of education, research, and development at the Seattle Proton Therapy Center. Dr. Dominello is an assistant professor in oncology department and practicing radiation oncologist at Wayne State University, Karmanos Cancer Center. His interests include stereotactic radiosurgery for brain and spine and therapeutic ratio. He currently serves as the Karmanos/McLaren‐wide PI for NRG, Karmanos Cancer Network Medical Director for Quality and participates as a member of numerous committees through NRG and ASTRO. Dr. Dominello serves as the Assistant Program Director for the Radiation Oncology Residency Program and as an instructor in courses for both graduate and undergraduate students at the university. Dr. Griffin is a professor of radiation biology at the University of Arkansas for Medical Sciences. His group studies living tissue response to high‐dose radiotherapy (SBRT) and spatially fractionated radiation approaches with targeted drug delivery to tumors. He served as a president of the Society for Thermal Medicine, is a Vice Chair of the Science Education and Professional Development Committee and Annual Meeting biology track chair for ASTRO and is an associate senior editor for Technology in Cancer Research and Treatment and the International Journal of Radiation Oncology, Biology and Physics.33.1Ion therapy was pioneered at Lawrence Berkeley National Laboratory (LBNL)CIRT has not been adopted in the United States because of cost and the absence of high‐level clinical evidence.3.2There are significant physical advantages of CIRT over photon and proton treatments.3.3Recent particle radiobiology studies indicate a number of potential underlying mechanisms for the promising phase I/II clinical outcomes with carbon ions.3.4The authors acknowledge that although the current clinical evidence supporting the use of carbon ion therapy is compelling, it is incomplete.3.5CIRT, compared with photons and protons, has the potential for superior dose distributions with enhanced biological effectiveness at a much lower cost than current facilities. Phase I/II trials suggest CIRT may well improve cancer outcomes. However, current high‐level phase III randomized clinical trial evidence does not exist.At least one CIRT facility is needed in the United States to (1) establish dose and fractionation regimens for US patients in different disease sites that are expected to benefit from CIRT; (2) conduct randomized clinical trials to establish which sites benefit from CIRT for the endpoints of therapeutic ratio, tumor control, treatment complications, and quality of life; (3) employ the best technology for the highest achievable accuracy and precision in targeting; (4) establish cost reductions through technology , amortization (decades), and patient load (reduced number of fractions); and (5) allow further investigations on the radiobiology of CIRT to elucidate tumor‐specific (epi)‐genomic determinants and tumor microenvironmental and immune‐modulatory effects that govern response and resistance.This carbon facility would pave the way to establish high‐level evidence for CIRT and would facilitate the technological innovation required to bring the cost down, advance treatment planning solutions, and improve the accuracy and reproducibility of treatment delivery.3.6Our consensus opinion about this topic is that, in light of the longstanding and ongoing struggles that many if not all proton centers have had, we are not in favor of the expansion of another ion beam therapy at great expense and questionable improvement in patient care. The following topics comprise the majority of the rationale and relevant literature in support of our assertion.It has been hypothesized that among patients undergoing radiotherapy, approximately 14–15% would potentially benefit from the dosimetric advantages afforded by proton therapy.https://www.nytimes.com/2018/04/27/business/proton-therapy-finances.html). While a single carbon facility in the United States may be able to attract many patients due to its novelty — which in large part will not be based on scientific evidence related to decreased side effects, new biology, or any other rational reason to pursue carbon ion therapy — it is not a viable modality to expand on top of the proton facility network already in existence, and already struggling. An additional obstacle beyond inadequate patient numbers for proton centers is the very real dilemma of insurance reimbursement. Some insurers will not reimburse for certain sites  while other centers get reimbursed at a rate that is not financially sustainable.Furthermore, despite acceptance of the potential advantage for protons in the select 14–15% of patients mentioned above, a number of proton centers have gone bankrupt and others struggle financially in part because they are not treating enough patients to meet their business model  building more centers. However, single‐center clinical trials are suboptimal sources of evidence.As a case in point, this is not necessarily a new topic that has never been considered for the United States. The United States had a heavy ion radiation therapy program at Lawrence Berkeley for many years between 1960s and 1990s.The primary argument for carbon beam is enhanced RBE. However, RBE is something of a double‐edge sword. Neutron therapy was an early example of a modality that delivers an enhanced RBE (3 or more). However, for the most part, neutron treatments used lower physical dose (by a factor of 3 or more) so that the net effect was the same as a photon beam. Only a few targets . Additionally, any CIRT facility within the United States would necessitate a location near a large population center, which would help ensure adequate patient numbers to maintain operations.In stark contrast to the early proton experience, the hard lessons learned in establishing proton therapy in the United States would help ensure more fiscally and scientifically sound efforts by the CIRT community. A new CIRT facility in the United States would be the center point for the design, implementation, and evaluation of a multi‐site, multi‐national effort of rationally designed clinical trials to establish the utility of CIRT. This would be modelled on the current successful collaborative clinical trial efforts with international CIRT sites. The US government could support R&D at the facility in the form of NCI‐sponsored clinical trials and grants to explore the engineering, physics, and biology of CIRT. The NCI has already demonstrated its support through funding of a pair of P20 planning grants to leverage CIRT R&D in a new facility, when it is built, and a trial of pancreatic cancer with a CIRT arm.The difficulty in convincing insurers to reimburse treatment is a common problem for new technology and one which should not dictate the next generation of radiotherapy techniques. Putting the patient first means implementing promising new technology that includes R&D to make it cost‐effective, guided by high‐level site‐specific evidence. We are not far from CIRT becoming cost‐effective: for chordoma, there are already published reports of this even with currently available technology.Here we address the “against sides” perception of the lack of strong support for a clinical replacement to the pioneering CIRT program at Berkeley. The promising results from BerkeleyWe agree that there are advantages to establishing multiple centers, but this does not rule out starting with a single center. New technology must be implemented in at least one center to establish its effectiveness. Since it will take longer to accrue patients for clinical trials with only one center, we need to start now. The first several proton centers established in the United States were instrumental in leading to additional centers, as will be the case with CIRT.Experience with neutrons was largely seen as a negative. In fact, the neutron RBE is quite effective, although the physics is suboptimal for deeper lesions, and the need for boron drug specificity for tumor has been a limitation for boron neutron capture therapy (BNCT). However, new accelerator‐based sources of neutrons are being evaluated in clinical trials, and despite the problems, BNCT experts in Finland are curing resistant tumors.Several issues with the current state of CIRT technology were considered as arguments against the proposal, but the reverse is true. A US CIRT treatment and research center will lead to rapid and cost‐effective technology advances. For example, range uncertainty mitigation and motion management are solvable problems. Although an SOBP has lower RBE than the Bragg peak, CIRT will still have a higher peak‐to‐plateau ratio in RBE‐weighted dose than protons. Regarding complexities of beam precision, one can always smudge the dose distribution, but one cannot improve intrinsic beam precision, an undeniable advantage of carbon over protons.Directing investment away from CIRT into potential "game‐changing" new investigations as the “against side” proposes is counter‐productive. Emerging technologies such as immunotherapy, MR‐linked therapy,The following is a point‐by‐point rebuttal to the position of the “against side.”Finally, we review recent outcomes of patients with adenoid cystic carcinoma (ACC) to raise the provocative question of how much data are needed to accept the utility of CIRT. Preclinical radiobiology studies and clinical investigations with charged particles have demonstrated favorable outcomes compared to photons, particularly with regard to toxicities, of this rare, yet highly aggressive neoplasm. Based on these early studies, photon therapy was combined with protons, resulting in 5‐year disease‐free survival and overall survival (OS) rates of 56% and 77%, respectively.4.2Drs. Faddegon, Tinkle, and Blakely have done an excellent job describing the status of carbon ion radiation therapy (CIRT). Unfortunately, the strongest argument against building such a facility comes from their introduction. As they state, much of the research has been done a long time ago (1967) at LBNL. As they themselves point out, that program was stopped due to cost, which remains just as much an issue today as it was then. As we have already stated, proton facilities struggle with cost issues, and our opponents state as much in their opening argument.They also point out the absence of high‐level clinical evidence for CIRT. More accurately, it is a lack of high‐level compelling evidence even after treatment of over 27,000 CIRT patients as of 2017.Our opponents also mention the current developments in proton/particle beam therapy (pencil beam scanning and reduction in range uncertainty). Certainly, these would be utilized in any new CIRT, but just as in proton therapy, they will only help CIRT keep up with the many advances in x‐ray therapy. They are not the silver bullet.Indeed, as our opponents show there has been a great deal of study of the radiobiologic effects of ion therapy. Unfortunately, promising in‐vivo research results often do not translate to the hoped‐for clinical results. Our colleagues have already admitted that there is a lack of high‐level evidence for CIRT. Radiobiology studies would be useful to determine how to best exploit CIRT. These studies are not a reason to build a CIRT. One needs more evidence than that. Although the radiobiology of carbon ions in tumor suggests that improved tumor control is possible compared to photons, the lack of increase in therapeutic ratio is the major hurdle that has not been met. The increasing ability of linear accelerators to conformally treat in 3D at very high doses and low normal tissue doses, combined with new developments suggesting that photons can be used to obtain an even greater therapeutic index using ultra‐high‐dose rates (FLASH), means that carbon ions will have a very hard time justifying their use.It follows that the clinical evidence cited by our opponents is weak, even as they describe it. The early US data are not a clinical trial. The Japanese and German data are only phase I/II trials; no phase III trials are completed. Furthermore, our opponents mention the results are “encouraging” for “tumors which are often resistant to conventional photon irradiation.” No advantage over more affordable proton therapy is suggested let alone shown. And finally, the authors point out that one cannot draw conclusions from trials which exclude some populations as the response can vary depending on the population studied.In their conclusion, the authors state that CIRT would have a superior dose distribution compared to protons. We disagree. Carbon ions undergo fragmentation, sending dose beyond the Bragg peak. The Bragg peak itself is positioned beyond the tumor to ensure tumor coverage putting the highest LET and highest RBE in normal tissue which, as mentioned above, will remain the greatest obstacle and practical barrier to obtaining improved patient outcomes. Protons clearly have a superior dose distribution over photons yet still struggle to show a clinical advantage. The difference between the carbon dose distribution and that of protons is much smaller by comparison so one cannot imagine it will have a markedly different or significant impact.Finally, the authors argue that a CIRT will employ the best technology, have a decades long life (to reduce cost), and use a reduced number of fractions. These are incompatible objectives. To show any advantage over current treatments, the best technology will have to be updated continuously. It will not be the best technology in 5 yr let alone 10 or 20 or 30 yr. And the ideal fractionation has already been stated as one of the goals of CIRT investigation, yet they realize they will be limited to hypofractionation for cost reduction. In conclusion, a single CIRT facility in the United States is unlikely to establish high‐level compelling evidence of a viable advantage over existing treatment options and we maintain that it will not be a worthwhile investment in this country for the foreseeable future.The authors declare no conflicts of interest.The views and opinions of authors expressed herein do not necessarily state or reflect those of US Government or any agency thereof or the Regents of the University of California.


None of the saturation mutant replicons significantly affected RNA replication. Notably, mutants encompassing the Walker motifs replicated as wild-type, showing indispensability of nucleotides conservation in viability compared to known criticality of amino acids. A 3D modeling of HEV-helicase and screening of a compound dataset identified ten most promising inhibitors with drug likeness, notably, JFD02650, RDR03130, and HTS11136 that interacted with Walker A residues Gly975, Gly978, Ser979, and Gly980. Our model building and virtual identification of novel helicase inhibitors warrant further studies towards developing anti-HEV drugs.The hepatitis E virus- (HEV-) helicase as a novel drug-target was evaluated. While cell culture model was used for mutational characterization of helicase, Orthohepevirus of the family Hepeviridae has at least seven genotypes (HEV1-HEV7), of which four (HEV1-HEV4) are known to infect humans [ in silico polyprotein sequence analysis of genetically-close viruses, methyltransferase (MeT/MTase), Y (undefined), papain-like cysteine protease (PCP), proline-rich hinge/hypervariable region (PPR/HVR), X (undefined/macro), helicase (hel/NTPase), and RNA-dependent RNA polymerase (RdRp) domains had been proposed in HEV ORF1 [The hepatitis E virus (HEV) is an emerging pathogen that causes acute hepatitis E in general and chronic infection in immunocompromised individuals , 2. Globt humans . Comparet humans . The HEV HEV1-HEV, of whict humans . Based oHEV ORF1 . FurtherHEV ORF1 , 11, PCPHEV ORF1 , HVR [13HEV ORF1 , X [15, HEV ORF1 , RdRp [1HEV ORF1 , 18, andHEV ORF1  domains 2+) binding activity, respectively [γ-phosphatase activity, probably involved in the first step of 5′ RNA capping in vitro [The HEV-hel/NTPase sequences  are mapped between X and RdRp domains [ectively . It has in vitro . In addiin vitro . Neverthα (pegIFN-α-2a) and ribavirin (RBV) have become the off-label drugs of choice [Because of self-limiting acute manifestation in general population, there has been no established treatment for hepatitis E. However, with the recent emergence of chronic infections, interferon-f choice –25. Thouf choice , 27. The pSK-GFP-WT  and pSK-GFP-GAD (RdRp mutant) were gifted by Dr. Suzanne Emerson . A series of ten consecutive saturation mutants  that completely covered the HEV-hel domain , appropriate amounts of mutant primers, dNTPs mix, DNA polymerase, and buffer under prescribed thermal conditions . The amplicons were Dpn I  digested, transformed (heat-shock) into DH5α XL-blue competent cells , and selected on ampicillin-agar plates. Plasmids  were confirmed for induced mutations by sequencing  and DNA stocks  were stored (-20°C).The green-fluorescence protein (GFP) reporter HEV repliconsl domain  were con2 supply. S10-3 cells (0.5 x106/well) were seeded in 24-well flat-bottom plates for transfection experiments.S10-3, the human hepatoma cell line , was cultured in complete DMEM media as described elsewhere  at 37°C  in vitro transcribed as capped genomic RNA and transfected into S10-3 cells, essentially as described elsewhere [ pSK-GFP-WT and pSK-GFP-GAD transfected cells served as positive and negative control, respectively. All transfections were done in duplicate and repeated twice.All replicons werelsewhere . Transfeμl/well, each) and recollected by rinsing the well with another 200 μl of PBS. Each culture (in duplicate) was pelleted at 4°C, resuspended in 300 μl of cold PBS, and immediately subjected to flow cytometry  and data were analyzed for GFP positive cells.The transfected cells were monitored on days 2, 4, and 6 for GFP production, the indicator of RNA replication under fluorescence microscope (Nikon H600L). The transfected cells were harvested using trypsin  on day 6 and processed as described previously . BrieflyThe HEV-hel  was modeled (corresponding to a.a. 1-232) using Swiss-modeler . By usinThe molecular docking tools AutoDock Vina  and Gene X-Score, a consensus scoring function was used in order to carry out docking validation [Thelidation . It uses in vitro synthesized RNA (data not shown). When transfected into S10-3 cells, none of the mutant transcripts significantly affected RNA replication ( pSK-GFP-hel7 mutant (nts. 3335-3406) had a mild downregulation of replication as compared to the wild-type (pSK-GFP-WT). Notably, the pSK-GFP-hel1 (nts. 2903-2974) and pSK-GFP-hel3 (nts. 3047-3118) mutants encompassing the Walker A and Walker B motifs replicated close to the wild-type. In addition, our replicon pSK-GFP-hel4 (nts. 3119-3190) with the mutant codons of naturally detected L1110 and V1120 substitution also had no effect on RNA replication  did not affect the gross yield oflication . Howeverlication . This vet nts. 33-3406 hadhttps://www.ebi.ac.uk/thornton-srv/software/PROCHECK/). It showed that while the phi-psi angles of 85.8% of the HEV-hel residues were in favored regions, 11.2% were in additionally allowed regions, 0.5% were in generously allowed regions, and 2.5% in the disallowed regions. The helicase model was also checked in Prosaweb server [The ToMV-helicase with best matched identity (33%) served as the suitable template for HEV-hel modeling. The modeled HEV-hel protein (residues no. 1-232 corresponding to HEV1 a.a. 963-1194) was validated by drawing Ramachandran plot (b server . The verThe stability and properties of the structure of the homolog were studied by explicit solvent molecular dynamic (MD) simulation. The root mean square deviation (RMSD) analysis not only reflects the change of protein backbone versus simulation time but also indicates the divergence of two structures. The RMSD of homolog became stable at 10 ns. The RMSD value of the modeled HEV-helicase was 0.4 nm . This reTo identify the best inhibitor molecules, a database of ~14000 compounds (selected from 56000 compounds dataset) were docked in the active site of HEV-hel using the AutoDock Vina and GOLD docking programs. The docked compounds were ranked based on their highest binding energy and GOLD Fitness Score with the corresponding protein. Finally, the top 10 ranked hits with higher binding and GOLD Fitness Score were considered and investigated further for their mode of interaction with the crucial residues (no. 1-232 corresponding to HEV a.a. 963-1194) in the modeled protein. The selected compounds  with theα-2a and RBV have become the antiviral drugs of choice [ in vitro replicon-cell culture model, followed by in silico protein modeling, molecular simulation, helicase-inhibitors screening, and drug likeness studies. Of a series of ten mutant replicons tested, none significantly affected RNA replication, showing the indispensability of HEV-hel nucleotides on virus replication. In contrast, the HEV RNA replication was abolished upon introducing amino acid mutations in both Walker A and Walker B motifs [ E. coli lost its enzymatic activities in vitro [ in vitro study, deletions in the HEV-hel Ia and III motifs significantly impaired ATPase and unwinding activities [In the current age of chronic HEV infections , pegIFN-f choice –25. Howef choice , 27. TheB motifs . Moreovein vitro . Notablyin vitro  are showin vitro . Contrartivities . Taken tBecause HEV1 and HEV2 are very pathogenic compared to HEV3 and HEV4 , the HEVOur saturation mutation analysis along with the helicase protein expression data strongly endorses the enzymatic function of HEV-hel domain. Moreover, the modeled 3D structure of HEV-hel enables virtual identification of ten most promising helicase inhibitors, notably, JFD02650, RDR03130, and HTS11136 with drug likeness properties that warrant further studies towards developing novel anti-HEV drugs.


RNA editing alters the identity of nucleotides in an RNA sequence so that the mature transcript differs from the template defined in the genome. This process has been observed in chloroplasts and mitochondria of both seed and early land plants. However, the frequency of RNA editing in plant mitochondria ranges from zero to thousands of editing sites. To date, analyses of RNA editing in mitochondria of early land plants have been conducted on a small number of genes or mitochondrial genomes of a single species. This study provides an overview of the mitogenomic RNA editing potential of the main lineages of these two groups of early land plants by predicting the RNA editing sites of 33 mitochondrial genes of 37 species of liverworts and mosses. For the purpose of the research, we newly assembled seven mitochondrial genomes of liverworts. The total number of liverwort genera with known complete mitogenome sequences has doubled and, as a result, the available complete mitogenome sequences now span almost all orders of liverworts. The RNA editing site predictions revealed that C-to-U RNA editing in liverworts and mosses is group-specific. This is especially evident in the case of liverwort lineages. The average level of C-to-U RNA editing appears to be over three times higher in liverworts than in mosses, while the C-to-U editing frequency of the majority of genes seems to be consistent for each gene across bryophytes. RNA editing is a modification of transcripts encoded by organellar and nuclear genomes that occurs in various organisms, including animals, plants, fungi, and protists ,2,3,4. TThe RNA editing process is known in most currently living plant lineages ,18,19, iThe frequency of RNA editing is poorly explored in the context of plant evolutionary diversification. In the plastomes of angiosperms, the RNA editing seems to decrease during diversification  and sevenad genes  revealed a decrease in RNA editing frequency during diversification of liverworts and mosses [Due to limited data availability, the evolutionary patterns of RNA editing in the mitogenomes of early land plants are poorly explored. The analysis of three mitochondrial d mosses . In the d mosses ,19. Mored mosses ,22, althd mosses .The backbone mitophylogeny of mosses and liverworts allows for phylogenetic insight into the evolution of quantity and type (C-to-U or U-to-C) of mitochondrial RNA editing. Earlier studies revealed mixed patterns of this phenomenon in bryophytes, but the datasets were usually very limited in terms of studied genes .The standard procedure used in the discovery of RNA editing sites is direct comparison of the RNA transcript sequence, usually obtained via RT-PCR or RNA-Seq methods, with genomic DNA sequence . HoweverIn this study, the predicted RNA editing patterns of 33 mitochondrial genes from 36 bryophytes were compared, comprising 18 liverworts and 18 mosses , represeFossombronia foveolata, Haplomitrium hookeri, Jungermannia sphaerocarpa, Metzgeria furcata, Porella platyphylla, Ptilidium ciliare, and Riccia fluitans  to 185,621 bp (R. fluitans), and the overall GC content varied from 44.8% (J. sphaerocarpa and P. ciliare) to 47.7% (H. hookeri)  . Both minad7). However, as in the previously studied species of Halpomitriopsida clade (Treubia lacunosa and Haplomitrium mnioides) [nad7 gene was also identified as functional, containing three exons, in H. hookeri. Other differences in gene content are due to lack of a nad7 pseudogene (J. sphaerocarpa), trnR-UCG gene (H. hookeri), and a second copy of the trnMf-CAU gene (F. foveolata and J. sphaerocarpa).The mitochondrial genomes of the seven species encoded 72–74 genes, including an identical set of 42 protein-coding genes, 27–28 tRNA genes, 3 rRNA genes, and 1 pseudogene (nioides) , the nadF. foveolata) to 30 (H. hookeri and R. fluitans). Although the mitogenomes of the seven liverworts species contain over a dozen of genes consisting of introns, the discrepancy in the overall intron number between species results from just four genes: atp1, cox1, nad7, and rrn18. The gene order of the seven species’ mitogenomes is similar to the other aforementioned species of liverworts [The number of genes containing introns ranges from 14 to 16 and the total number of introns within the mitogenomes varied from 25  and two complete mitogenomic sequences (Fossombronia foveolata and Metzgeria furcata) of simple thalloid provide a great extent of the sequence data of this poorly explored  group of liverworts. To date, the only representative of simple thalloid liverworts with a completely known mitogenome sequence was Aneura pinguis [Seven mitochondrial genomes obtained in this study double the total number of liverwort genera with known complete mitogenome sequences. Moreover, two sets of mitochondrial protein-coding sequences  and 30 were found in B. pusilla (17 C-to-U and 13 U-to-C). In both mitogenomes, the share of reverse U-to-C editing sites in total number was high, unlike in other known mitogenomes of liverworts and angiosperms [For several genes, a significant drop in predicted editing sites appears in specific taxonomic groups. Besides the known, non-editing model liverwort genus rchantia , two othiosperms ,42. HoweThe total number of predicted RNA editing sites declined in the leafy liverworts, which are a late-diverging group .Calypogeia and Tritomaria mitochondrial genes with other known liverwort mitogenomes revealed precise intron loss [The trend of decreasing number of predicted editing sites along with diversification observed in the leafy liverworts group could be connected with structural changes of the mitochondrial genes, i.e., intron losses ,40. The ron loss ,44. It iron loss , but somron loss ,46,47,48Treubia lacunosa, while a sister to this species, Haplomitrium hookeri, was the richest in the editing site species with a total of 732, including 712 C-to-U and 20 U-to-C sites. This was also confirmed in the previous studies on nad4 and nad5 genes [Haplomitrium mnioides, suggesting a comparable level of RNA editing as predicted in H. hookeri.However, not all species and genes fit into the above scenario. A low number of editing sites (89) were detected in the early divergent liverwort, d5 genes , where 5The C-to-U editing frequency, expressed as the number of predicted C-to-U editing sites per gene CDS length, was calculated for each of the 33 protein-coding genes of 17 species of liverworts (see method section for details). The U-to-C editing frequency was calculated analogously. For every liverwort group , the average editing frequencies were calculated and defined as mean C-to-U or U-to-C editing frequencies of each gene . Howeverrps13 to 2.17% in atp9 and from 0.32% in rps12 to 4.98% in atp9 in leafy and simple thalloid liverworts, respectively  differences between the means of C-to-U editing frequency in mitogenomes of simple thalloids and leafy liverworts , the number of predicted editing sites was relatively low and stable among analyzed mitogenomes of leafy and simple thalloid liverworts. The most notable decrease (3-fold or more) in editing frequency in leafy compared to simple thalloid liverworts was found in atp6, cox3, nad4, sdh4, cox1, rpl6, and rps4 genes  differences between means of U-to-C editing frequency in the mitogenomes of simple and leafy liverworts   did not reveal significant  . HoweverPtychomnion cygnisetum, the representative of plagiotropic mosses, also contained the most C-to-U predicted editing sites (124) of all studied moss species. In contrast, Funaria hygrometrica mitogenome, with just 9 C-to-U and 5 U-to-C predicted editing sites, was the least editing-rich species . For every moss group , the average editing frequencies were calculated and defined as mean C-to-U or U-to-C editing frequencies of each gene . rpl16 and nad4L to 1.32% in tatC, from 0% in atp4, nad2, nad4L, rpl16, rps12, rps13, and rps19 to 0.73% in atp9 and from 0% in rps2 to 0.96% in atp9 in early-diverging, orthotropic, and plagiotropic mosses, respectively  between at least two groups of mosses in 20 out of 33 protein-coding genes. Hence, the mosses seem to be a more homogenic group of plants, in terms of RNA editing, than liverworts, in which 31 protein-coding genes varied significantly  between at least two groups in predicted C-to-U substitutions  across genes. A visual inspection of scaled means  of gene C-to-U editing frequencies  between a C-to-U editing frequency of 33 genes of liverwort and moss mitochondria. This suggests that, regardless the overall RNA editing level, the frequency of C-to-U RNA editing is preserved across genes of bryophytes. Although the majority of mitochondrial genes investigated in this study seem to be in accordance with this hypothesis, for some genes, like nad2, nad4L, nad6, rps7, and rps14, RNA editing seems to be taxon-dependent. Research focusing on the mitochondrial frequency of editing sites across phylogeny has not yet provided an unambiguous answer. A recent paper on 17 diverse angiosperms [atp9 gene displays a high potential of C-to-U RNA editing frequency in bryophytes, whereas no editing sites were identified in the atp9 of angiosperms [The current study revealed the moderate overall positive correlation , the character-state matrix analysis could not be performed as well as for editing-site rich taxa. The second incongruence is the position of leafy Pleurozia purpurea. This taxon in the earlier studies was placed as a sister to Metzgeriales [Pleurozia purpurea within the leafy liverworts clade with good bootstrap values (84% maximum parsimony and 100% neighbor-joining bootstrap support) and provided result congruent with morphology. However, to provide better support for the leafy liverworts monophylly further investigation with the use of plastid and nuclear genes is required.The alignment of the set of 33 protein-coding sequences of mitochondrial genes common to liverworts and mosses   was usedlytomies B. Phyloglytomies B, mainlygeriales ,66 and iTotal genomic DNA was extracted using ZR Plant/Seed DNA MiniPrepTM kit  according to the manufacturer’s protocol from fresh tissue or herbarium specimens. The extracted DNA was quantified using a Qubit BR DNA kit . The genomic libraries were prepared using a TruSeq Nano DNA library preparation kit  and sequenced using HiSeqX sequencer (Illumina) by Macrogen Inc.  to generate 150 bp paired-end reads with a 350 bp insert size between paired-ends. The list of GenBank accession numbers and newly sequenced species are given in Sequencing reads were cleaned by removing the adaptor sequences and low-quality reads with Trimmomatic 0.36  . The filPellia epiphylla and Pallavicinia lyellii, only complete CDS datasets were obtained, since the reliable assembly of complete mitogenomes was difficult due to the presence of tandem repeat-rich regions.The newly sequenced mitogenomes were annotated as described in the authors’ previous studies on liverworts ,39,40. IPhyscomitrella patens—11 RNA editing sites identified) or no  evidence of RNA editing, which is desired while predicting RNA editing events. The M. paleacea mitogenome was used as a reference mitogenome for the liverwort CDS set analysis while the P. patens mitogenome was used as the reference mitogenome for the moss CDS set analysis. The prediction of RNA editing sites produced 35 strings of numbers indicating the positions of single bases within 33 genes . Next, based on the strings, the number of RNA editing sites predicted in each gene of every species was calculated  and three mosses . The transcriptome datasets were mapped to 33 corresponding protein-coding sequences of mitochondrial genes using Geneious 8.1 software  [The 1000 plants project (1KP)  data wasZealand) . The obtZealand)  and the Zealand)    and MAFFM. paleacea and P. patens could not be included in this analysis since they were used as references in RNA editing sites predictions. First, since RNA editing sites of each species were predicted relatively to the same set of reference genes, all C-to-U editing positions identified within each gene across species were summarized as an RNA editing template of a gene. Next, the gene templates were concatenated in order to obtain one template for a whole set of genes. Afterwards, RNA editing positions of each species were mapped to the template to obtain a binary matrix of RNA editing predictions. The liverworts’ matrix contained a total of 1285 unique predicted editing sites positions, while the mosses’ matrix contained 435 . Finally, based on a binary matrix of RNA editing converted to NEXUS file format, the Bayesian inference   and MrBayes 3.2.1  [H. hookeri as an outgroup, while the mosses’ tree was depicted with S. palustre as an outgroup  , maximumNY, USA) . The livoutgroup .p-value cut-off < 0.05) was used to compare the C-to-U and U-to-C means of editing frequency of genes among three groups of moss as well as three groups of liverwort. P. patens was excluded from the analysis, unlike M. paleacea (confirmed non-editing mitogenome), since previous studies reported 11 editing sites identified within its mitogenome. The post-hoc Dunn’s test with the Benjamini-Hochberg p-value adjustment method  was used to pinpoint which means of editing frequencies were significantly different from the others. The Pearson correlation coefficient was applied to measure the correlation between means of C-to-U editing frequency. All statistical analyses were conducted using the R software environment  and PMCMR software package [The Kruskal–Wallis test ( package .H. hookeri liverwort (712 C-to-U editing sites) and P. cygnisetum moss (124 C-to-U editing sites). The average mitochondrial C-to-U RNA editing level was also over three times higher in liverworts than in mosses. Despite considerable differences in editing level, the C-to-U editing frequency of majority of genes seems to be similar for each gene across the two groups of early land plants. Such genes as atp9, sdh3, or tatC showed high, while atp8, rpl16, and rps12 showed low C-to-U editing frequency, both in mosses and liverworts relatively to the overall predicted C-to-U editing level. Contrarily, the U-to-C RNA editing site predictions did not reveal significant differences in editing frequency across taxonomic groups of either liverworts or mosses. However, the validation of U-to-C RNA editing predictions using database-derived transcriptomes suggests that the U-to-C prediction models require further improvement.To obtain a comprehensive view of the RNA editing potential of mitogenomes of bryophytes, 37 sets of 33 mitochondrial protein-coding sequences were analyzed. The results obtained in this study concerned the main lineages of liverworts and mosses, which is the first case of such extensive study of mitogenomic RNA editing in early land plants. For the purpose of this research, the number of assembled liverwort mitochondrial genomes was expanded by seven and additionally provided two nearly complete sets of mitochondrial genes of liverworts. The newly-assembled mitogenomes display features typical of liverworts. The predictions of RNA editing sites within whole set of mitogenomes of 17 liverworts and 18 mosses species revealed that the level of C-to-U RNA editing in liverworts and mosses is group-specific. Among all investigated species, the highest number of editing sites was predicted in The analyses of RNA editing patterns demonstrated that RNA editing potential hidden in the mitochondrial genomes of liverworts and mosses is widespread and may have considerable impact on mature transcripts encoded by mitochondrial DNA, especially among liverworts.


Influenza virus causes seasonal epidemics and dangerous pandemic outbreaks. It is a single stranded (−)RNA virus with a segmented genome. Eight segments of genomic viral RNA (vRNA) form the virion, which are then transcribed and replicated in host cells. The secondary structure of vRNA is an important regulator of virus biology and can be a target for finding new therapeutics. In this paper, the secondary structure of segment 5 vRNA is determined based on chemical mapping data, free energy minimization and structure-sequence conservation analysis for type A influenza. The revealed secondary structure has circular folding with a previously reported panhandle motif and distinct novel domains. Conservations of base pairs is 87% on average with many structural motifs that are highly conserved. Isoenergetic microarray mapping was used to additionally validate secondary structure and to discover regions that easy bind short oligonucleotides. Antisense oligonucleotides, which were designed based on modeled secondary structure and microarray mapping, inhibit influenza A virus proliferation in MDCK cells. The most potent oligonucleotides lowered virus titer by ~90%. These results define universal for type A structured regions that could be important for virus function, as well as new targets for antisense therapeutics. Currently, there are only a few treatments or protections available against influenza. Vaccines, containing inactivated or attenuated virus or pure viral proteins must be reformulated and administered annually; efficacy levels can vary greatly depending on what strains actually circulate3. The currently available drugs target viral surface proteins: these include M2 ion channel blockers and neuraminidase inhibitors4. However, resistance to small-molecule therapeutics is growing4. There is a great need to find other approaches for combating influenza virus. One promising new approach focuses on influenza RNA as a therapeutic target.Influenza virus causes global epidemics each year and infrequent, but dangerous pandemic outbreaks. According to the WHO, seasonal flu is responsible for up to 650000 deaths yearly5; which, in virions, exist in viral ribonucleoprotein (vRNP) complexes that contains (beside vRNAs) multiple NP and polymerase proteins. Each vRNP complex is an independent replication-transcription unit that uses its heterotrimeric viral RNA-dependent RNA polymerase. vRNA is a template for mRNA and complementary (c)RNA that is used as a template for replicating vRNA7. RNA is used throughout the viral replication cycle, highlighting its central role in influenza biology. Cryo-electron microscopy has provided important insights into the global 3D vRNP structure9. Later, using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), the vRNA binding profiles of NP was identified for two strains10. These studies revealed that vRNA can loop out from complex with NP and potentially form secondary structure, even in RNP. In cells, viral RNA structure could have additional roles depending on the step of the replication cycle.Influenza is an RNA virus with a single stranded, negative-sense viral genome (v)RNA. vRNA is divided to eight segments13. Multiple structured regions have been confirmed experimentally17 and several were confirmed as functional by additional experiments18. So far, only the secondary structures of segments 7 and 8 vRNAs were experimentally determined in vitro (the full genome sequence)20. Knowledge of vRNA secondary structure is crucial for understanding virulence and developing inhibitors; thus, in this study, the secondary structure of the segment 5 vRNA (vRNA5) was modeled. This segment encodes NP protein, which plays several roles during the viral life cycle, including vRNP formation, transport of vRNA-polymerase complex to the nucleus, viral replication, and virion packaging5. Because NP protein is abundant—compared to other viral proteins—and appears in almost each step of virus propagation, it draws the attention of scientists both in terms of understanding its functions and as a potential target of new therapies. Recently, several RNA structural motifs were proposed for the vRNA521. Other studies of vRNA5 indicated that select regions interact more weakly with NP, therefore allowing them to fold into RNA motifs22. The functions of vRNA5 motifs were verified by mutagenesis: e.g. synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect22. NP binding profiles also confirm previous results and showed that there are differences NP associations between strains24. This interesting finding could explain different patterns of interactions between vRNPs through RNA found by others. Recent data reveals highly structured genomes with space to allow a redundant network of vRNA-vRNA interactions necessary for packaging25.RNA function is closely related with its structure. For influenza virus structure-function relationships are emerging as new studies continue to discover roles for RNA structure. Initial bioinformatics scans revealed the possibility of finding conserved structural motifs across both (−) and (+) strandsHerein, the secondary structure of entire segment 5 vRNA (vRNA5) is determined for the first time. The basis of prediction was chemical mapping supported by isoenergetic microarray mapping, free energy minimization and bioinformatics analysis. The data presented complements previous results regarding the suggestion of existence of vRNA5 structural motifs by presenting an experimentally-informed global view of whole segment structure, which shows conservation across a variety of strains. Furthermore, results showing oligonucleotide accessibility can act as leads for designing antisense oligonucleotides: indeed: several oligonucleotides are shown to be active vs. vRNA5 and inhibit virus proliferation.28. Before mapping, appropriate folding conditions were optimized to yield a single RNA conformation, as judged by non-denaturing agarose gel , CMCT methylates N3 of U and kethoxal methylates G at N1 and N2gel Fig. . Resultsgel Fig. .Figure 1Materials and Methods). The vRNA5 structural model with the lowest Gibbs free energy was selected as the final model structure  are in domain II. There are 23 hairpins present in the vRNA5 secondary structure overall. Some of them are strongly mapped with modifying reagents in loop regions: e.g. nts 87–115, 493–502, 409–418, 1074–1115, 1136–1160, 1404–1429 and 1483–1497. The longest helix motif (811–871/1017–1064 nt) was identified in domain III and it is compatible with the few modifications observed in this region  with 877 different penta- and hexanucleotides probes were used , which is the largest region in vRNA5 not accessible to probes. All RNA fragments that do not bind to isoenergetic probes are marked on Fig. Prior to microarray mapping, vRNA5 was radioactively labeled during NA5 Figs . Some prAn RNAse H assay was used as additional approach for the estimation of vRNA5 accessibility to oligonucleotides in selected regions and to clarify interpretation of microarray mapping results for these regions. In this study, 14 DNA oligonucleotides were designed Table  to evaluAll RNase H cleavage sites (strong and weak) in the vRNA5 secondary structure are presented on Fig. The base pair probabilities of the vRNA5 secondary structure model were estimated as described in the Materials and Methods; single stranded nucleotide probabilities were also estimated. In the calculation, experimental structure mapping data were incorporated as constrains. The calculated probability data give information about structure prediction quality and identify well-defined structural motifs. Results are presented on Fig. . The mosMaterial and Methods; Supplementary Data Structure conservation of the proposed secondary structure model was analyzed using all available influenza A sequences  was highly conserved (97.5%). In addition to the panhandle structure, ca.130 nt from 5′ and ca. 100 nt from 3′ end of the vRNA5 sequence also encodes packaging signals34. Interestingly, conserved hairpins were found in both regions: spanning nts 87–115 and 1483–1497. Both hairpins are conserved . Variations in sequence could preserve base pairing (particularly in nts 87–115), however, numerous inconsistent mutations were observed in the less well conserved (latter) hairpin  for base pairs 1084/1114 nt-1088/1099 nt. The vRNA5 base pair conservation of 86.8% is calculated for helix 1117–1125/1211–1203 nt, whereas the structural motif in region 1127–1173 nt is conserved 84.4%. The conservation of 460–476 nt hairpin is 83% and for hairpin 477–484 nt is 95.6%   targeting vRNA5 were designed to inhibit influenza virus replication. Additional negative control oligonucleotides were used: NEG (ASO non-complementary to vRNA5)M) Table , Fig. 6 For selected ASOs, qRT-PCR was used to determine the levels of vRNA reduction. The results were compared with the standard curve and are presented in Fig. The most inhibitory ASOs were tested for cytotoxicity at concentrations of 0.5 μM using MTT assay. The ability of NAD(P)H-dependent cellular oxidoreductase enzymes to function is measured, this reflects cell viability Fig. . In genein vitro chemical mapping coupled with free energy minimization and bioinformatics sequence/structure analysis. vRNA5 is highly structured with distinguishing motifs and domains. Evidence of single stranded and accessible regions were showed by reactivity of nucleotides to four mapping reagents, binding to isoenergetic probes and targeted RNase H cleavage in the presence of selected DNA oligonucleotides  Fig. . The stret al. to be possible both in the (−) and (+) strand as a “mirror structures”21. Among those motifs, there is a hairpin at 1527–1550 nt (16–39nt in + RNA) which is located within the packaging signal, and it is possible that this motif may be important for packaging. The presence of the 1527–1550 nt hairpin in the vRNA5 structure is very conserved across strains of type A  can form this structure, our model of the secondary structure of this region is different, also the chemical mapping profile excludes the formation of this hairpin   Gultyaev 21. Secondary structure studies revealed that the potential pseudoknot base pairs (124–130/96–102 nt) had strong chemical mapping signals that preclude their formation in vitro RNA5 have several similar structural motifs: e.g. 1341–1454 nt (107–227 nt in (+)RNA), 89–113 nt (1453–1477 nt in (+)RNA), 460–476 nt (1090–1106 nt in (+)RNA, 1243–1273 nt (293–323 nt in (+)RNA), 1472–1506 nt (60–94 nt in (+)RNA), 1531–1545 nt (21–35 nt in (+)RNA) RNA; however, only vRNA segments are packaged into progeny virions, while cRNAs are degraded in the host cell. In the process of packaging, M1 binds only vRNP. cRNP is not recognized by M1 and, as a result, the transport of vRNP and cRNP in the cytosol differs37. Specific binding of M1 is responsible for packaging of vRNP into virions and unique features should be used. Structure similarity in 5′and 3′ ends regions could be needed for NP and polymerase binding to form vRNP and cRNP complexes. This suggest that not only sequence/secondary structure in the ~150 nt fragments at 5′ and 3′are important to packaging, but internal regions may also have an impact on the process. This is consistent with observations for other vRNA segments, where internal regions are responsible for vRNA/vRNA intermolecular interactions40.A secondary structure model of (+)RNA5 based on A/Vietnam/1203/2004 (H5N1) strain was determined previouslyNA) Fig. 36. Most In vitro experiments in native buffer with the absence of proteins allows one to study exclusively RNA properties and enables clear interpretation of results. Rigid and flexible regions of RNA structure, and the possibility of rearrangements could be important for different RNA functions. Also, they can be targets for future drugs which disturb or block RNA structural motifs.Knowledge about naked vRNA structure is needed to identify thermodynamically stable structure motifs that could preferentially form and have biological function. 42. In general, influenza can infect different host species and replicate over a large range of temperatures42. The temperature-stimulated differences observed in the panhandle are consistent with hypothesized functions connected to alternative folds of the 5′ end 3′ ends of the vRNA (such as the hook and cork screw conformations)44. Likewise, other dynamic regions could also be important in the virus replication cycle. For example, the rearrangements in domain II, which includes a packaging signal sequence may be functional.Lowering temperature to 23 °C causes rearrangements in mainly two parts of the structure, but overall the differences are small Fig. . This lo22. This allows for a vRNP structural model where vRNA loops out from the protein complex and folds to create functional structural motifs—possibly in a dynamic manner10. From the studies of two vRNA5 strains (A/WSN/1933 (H1N1) and A/California/07/2009 (H1N1)), similarities were noticed in NP binding profiles. There are also differences that are specific to each strain, especially in proportion of bound NP to different vRNA regions10. In general, in regions located within the promoter and packaging signals at the 3′ and 5′ ends, NP binding is reduced and the major NP binding sites can be identified within the internal region. Significant lowering of NP binding for both A/WSN/1933 (H1N1) and A/California/07/2009 (H1N1) were noticed in internal regions of vRNAs, such as around 330–490, 600–640, 750–950 and 1140–1400 nt, which could be a common feature10. These fragments in vRNA5 form local motifs or interact with other RNA region . It was shown, however, that NP monomers bind irregularly, which allows vRNA structural motifs to be formed45. Identified vRNA5 structural motifs, especially in low NP binding regions, could be crucial for packaging and/or replication.Low NP binding regions are accessible for inter- and intramolecular RNA interaction vital for the virus replication cycle. Indeed, it was shown in different studies that synonymous mutations that abolish predicted RNA secondary structure motifs result in attenuation of IAV replication48. Moreover, only the panhandle region and nearby 3′ end regions were targeted so far. Therefore, for the first time, interior regions of vRNA5 were effectively targeted. Also, modified RNA oligonucleotides (2′OMeRNA and 2′OMeRNA-LNA) were used here, confirming their usefulness in antisense strategies using a low dosage of oligonucleotide.The determined secondary structure of vRNA5 coupled with isoenergetic microarray mapping led to designing of effective antisense oligonucleotides. Few papers report antisense oligonucleotides that target vRNA5 and inhibit virus replicationMost ASOs influenced virus replication  have target region fragments found to have low in vivo10. Oligonucleotides 883–11L and 883–11M may affect a possible interaction between vRNA5 and vRNA2. Nucleotides 874–883 in vRNA5 are complementary to region 25–34 nt in vRNA2: the binding of 883–11M and 883–11L to nts 878–888 nt would preclude this putative vRNA-vRNA interaction. The highest inhibition, observed for 883–11L vs. 883–11M could come from more stable duplex with target RNA for LNA modified oligonucleotide.The most effective antisense oligonucleotide is 883–11L, which targets region 878–888 nt Figs –8. Interin vivo NP region, is located the target site for 474–21M and 474–21L oligonucleotides (465–485 nt)10. Surprisingly, higher inhibition was observed with 2′OMeRNA 474–21M (64%) than the same oligonucleotide containing LNA modifications 474–21L (31%)  Figs .10 antisense oligonucleotides 1253–13M, 1253–13L and 1333–16L  for combined treatments. Also, vRNA5 fragments accessible to oligonucleotides could be used for techniques applying complementary oligonucleotides: e.g. 50. To solid support was added 32% ammonia solution and incubated in 55 °C, 18 h, than evaporated and dissolved in water. Primers for reverse transcription, contained C6-aminolinker at 5′-end, were treated, in next step, with 80% acetic acid (3 h), precipitated in 1% sodium perchlorate solution in acetone and labelled with 5-FAM, 6-JOE, 5-ROX or 6-TAMRA (Anaspec). 300 µg of oligonucleotide was dissolved in water (11 µl) and 75 µl of 0.1M sodium tetraborate, pH 8.5 and fluorophore  in 14 µl DMSO was added. Reactions were incubated at 23 °C for 18 h by slowly mixing (150 rpm) then precipitated and run on 12% denaturing PAA gel. Oligonucleotides for ribonuclease H assay were purified using thin layer chromatography (TLC) and mobile phase was n-propanol/ammonia/water (52/35/13). Antisense oligonucleotides (2′-O-methyl RNA and 2′-O-methyl RNA containing LNA modifications (LNA - locked nucleic acids) were purified with TLC (short oligonucleotides) or denaturing 12% PAA gel (oligonucleotides longer than 11 nt). Oligonucleotides concentration was measured at 260 nm with Nanodrop spectrophotometer (Thermo Scientific). Molecular weights were confirmed by mass spectrometry .The synthesis of oligonucleotides was performed via MerMade 12 solid phase synthesis with phosphoramidites. All oligonucleotides  were deprotected according to published procedureTM T7-FlashTM Kit and then RNA was purified with RNeasy MinElute Cleanup Kit. The yield was measured with Nanodrop® spectrophotometer at 260 nm and quality of RNA was examined by agarose gel (1%) electrophoresis at 4 °C and with comparison with RiboRuler High Range RNA Ladder.DNA template of A/Vietnam 1203/2004 (H5N1) segment 5 vRNA (vRNA5) was obtained from the pHH21 plasmid. For PCR were used PR1 and forward PR2 (with T7 promoter sequence) Table . Transcr2, 50 mM HEPES pH 7.5) or buffer 2  at 65 °C for 5 min and cooled down to room temperature. Appropriate folding to one native structure was checked on 1% agarose gel  at least three datasets were obtained from each primer and the average of results was used for prediction of secondary structure.ABI files were analyzed with ShapeFinder software54 with incorporated experimental data55. From CMCT, DMS and kethoxal mapping data only reactivities ≥0.700 were used as constraints . At the same time NMIA reactivities from SHAPE mapping as pseudoenergies were implemented in prediction . The Slope and Intercept parameters were 1.8 and −0.6, respectively56.The secondary structure of vRNA5 was generated using RNAstructure 5.7 software52 program implemented in RNAstructure Web Server for RNA structure Prediction (Predict a Secondary Structure mode). The chemical mapping experiments results were incorporated and all parameters were as described in Secondary structure prediction section.The vRNA5 base pair probabilities were obtained using RNAstructure57. The coding RNAs were reverse transcribed in silico then aligned using MAFFT (FFT-NS-1 method)58. vRNA5 structure models were mapped to the alignments then preservation of base pairing was calculated to give the percent conservation of canonical base pairing  as well as a measure of inconsistent, or potentially non-canonical, pairs are model base pair sites. Swaps between canonical pairing types were manually inspected to identify potential structure-preserving (consistent or compensatory) changes.All available vRNA5 sequences were obtained from the NCBI Influenza Virus Resource60. Microarray slides  were coated with 2% agarose, reduced with NaIO4, washed in water and dried overnight. Probes for isoenergetic microarrays are 2′-O-methyl-LNA penta- and hexamers containing selected 2,6-diaminopurine riboside modification (LNA or 2′-O-methylated nucleotide)61. All probes comprising C6-aminolinker at 5′ and were printed in three repeats with NanoprintTM at 50% humidity. The final concentration of probes was 0.1 mM and printing buffer was 3xSSC, 0.05% SDS, 0.001% CHAPS. List of complementary probes to vRNA5 is in Supplementary Data 4 (35 mM) solution in ethanol and 1x PBS buffer (1:3 v/v). Then, isoenergetic microarrays were washed of reducing reagent in water (3 times) and dry.Isoenergetic microarrays were similarly as described previously62. For experiment vRNA5 was labelled with [α32P] ATP during in vitro transcription and purified with RNeasy MinElute Cleanup Kit. Upon every hybridization experiment vRNA5 was diluted in buffer 1 or buffer 2 and final concentrations of vRNA was 2 μM.Microarray mapping using isoenergetic microarrays was conducted similarly as described beforeRadioactively labelled vRNA5 was folded in selected buffer (buffer 1 or buffer 2) as described above. Hybridization was performed with 300 000 cpm vRNA5 on microarray slide at 100% humidity chamber. The final volume was 250 μl and incubation was performed for 18 h at both 37 °C and 23 °C. Then microarrays were washed (1 min and temperature of buffer was 37 °C), dried and after exposure to imaging screen scanned by Fujii phosphoimager.Quantitaive analysis were performed with ArrayGauge V2.1. Binding intensities were denoted as strong, medium and weak when the integrated spot intensity was ≥1/3, ≥1/9 and ≥1/27 of the strongest integrated spot intensity, respectively. The binding site was named from the number of RNA middle nucleotide, complementary to the probe sequence.vRNA5 (3 μM) was folded and next incubated with appropriate short DNA oligonucleotide (3 μM) in buffer 1 with 1 mM DTT, then 5U of ribonuclease H was added. The reaction was carried out at 37 °C for 30 min. The reaction was stopped by heating for 10 min at 65 °C. Then, precipitation and primer extension were performed as described above.2 humidified incubator. Influenza virus A/California/04/2009 (H1N1) was a gift from Prof. Luis Martinez-Sobrido, University of Rochester. Virus titers were determined with standard plaque assays64.MDCK were obtained from Sigma-Aldrich. Growth medium was DMEM with 10% fetal bovine serum (FBS), 1x PSG  and cells were cultured at 37 °C in a 5% CO6 cells were seeded in 10 cm2 plate. Prior to transfection 0.5 μM of each oligonucleotide was vacuum-dried, than dissolved in OPTIMEM® medium (Life Technologies). Transfection was carried out with Lipofectamine 2000 according to manufacture instruction. The experiment was performed with both 96-well and 24-well plates. In the case of 96-well plate experiment the final Lipofectamine 2000 concentration was 0.4 μl/well. During incubation of oligonucleotide with Lipofectamine 2000 cells were passaged and after 25 min they were added to OPTIMEM®-Lipofectamine-oligonucleotide mix. Cells with oligonucleotide were seeded on 96-well plate (2 × 104 cells/well) and after 12 h the medium was changed for the standard growth medium and incubated at 37 °C in a 5% CO2 humidified incubator for the following 6 h. For 24-well plate experiment all reagents were scaled up proportionally to 96-well plate experiment.24 h before experiment with antisense oligonucleotides 2.5 × 102 in a humidified incubator. Supernatants with virus were collected after 24 h. Virus titer of A/California/04/2009 (H1N1) was calculated with the Indirect Immunofluorescence Assay (IFA). Additionally, for selected oligonucleotides, qRT-PCR was used for calculation of vRNA level.Next, cells were infected with influenza virus. In details, 18 h after transfection MDCK cells were washed with 1x PBS  pH 7.4 and infection was conducted in infection medium ). MDCK were infected with wild-type virus A/California/04/2009 (H1N1) at MOI of 0.001. After 1 h of infection at room temperature medium of 0.3% BSA, 1x PSG, TPCK-trypsin 1 μg/ml in DMEM was added. Plates were incubated at 33 °C in a 5% CO96-well plates were infected with thawed supernatants in 10-fold dilutions. 8 h after infection the fix and permeabilization solution was added . Incubation was carried out for 15 min at room temperature. Then cells were washed with 1x PBS and blocking buffer was added (3% BSA in 1x PBS pH 7.4) and plates were incubated for 12 h at 4 °C. Blocking buffer was aspirated and solution of antibody  100x diluted in the blocking buffer was added (45 μl/well). After 2 h of incubation at 33 °C, the solution was aspirated, plates were washed 3x with 1x PBS and rabbit anti-mouse IgG antibody (1 mg/ml in the blocking buffer) with FITC (Merck Millipore) was added. Plates were incubated for 2 h, washed with 1x PBS, visualized under the fluorescence microscope and FFU/ml was calculated.65. Total RNA was isolated from infected cells. 24 h after infection (performed on 24-well plate) 500 μl of TRIZOL reagent was added on every well. RNA was isolated with Chomczynski Sacchi method protocol66. 500 ng of prepared probes were treated with 2U of DNase I for 30 min at 37 °C and RNA quality was estimated with agarose gel electrophoresis. Next, 2 μl of prepared RNA was used for reverse transcription with 1pmol of primer RT -2,5-diphenyltetrazolium bromide] assay was carried out. MDCK cells were seeded in 96-well plates  and were grown up to 80–90% confluency. Than transfection was conducted with 0.5 μM of oligonucleotide. The control was Lipofectamine 2000 solution. After 12 h MTT (5 mg/ml) in DMEM was added and plates were incubated for 2 h. The medium was removed and DMSO (100 µl per well) was added. Plates were put on the rocker (slow movement) for 15 min and absorbance was measured at 570 nm . Cell viability was presented as the percentage ratio of absorbance of oligonucleotide well to the absorbance of control well.Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3


The 5′-cap structure, characteristic for RNA polymerase II-transcribed RNAs, plays important roles in RNA metabolism. In humans, RNA cap formation includes post-transcriptional modification of the first transcribed nucleotide by RNA cap1 methyltransferase (CMTr1). Here, we report that CMTr1 activity is hindered towards RNA substrates with highly structured 5′ termini. We found that CMTr1 binds ATP-dependent RNA DHX15 helicase and that this interaction, mediated by the G-patch domain of CMTr1, has an advantageous effect on CMTr1 activity towards highly structured RNA substrates. The effect of DHX15 helicase activity is consistent with the strength of the secondary structure that has to be removed for CMTr1 to access the 5′-terminal residues in a single-stranded conformation. This is, to our knowledge, the first demonstration of the involvement of DHX15 in post-transcriptional RNA modification, and the first example of a molecular process in which DHX15 directly affects the activity of another enzyme. Our findings suggest a new mechanism underlying the regulatory role of DHX15 in the RNA capping process. RNAs with highly structured 5′ termini constitute a significant fraction of the human transcriptome. Hence, CMTr1–DHX15 cooperation is likely to be important for the metabolism of RNA polymerase II-transcribed RNAs.This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’. Reactions were carried out with 2.4 pmol of purified CMTr1 enzyme and 4.8 pmol of substrate RNA, in a total volume of 30 µl, at 37°C, for 1 h or the time indicated. Samples were applied on the Nylon membrane, dried and washed with 200 mM phosphate buffer (pH 6.5) to remove free [3H-methyl]-SAM. Then, samples were incubated for 30 min in scintillation liquid in the dark, and the measurements were performed by a scintillation counter .Methyltransferase activity assay was performed in BDHX buffer . Mixtures containing both enzymes were incubated on ice for 10 min, before addition of the RNA substrate and SAM, and processed further as described above. Complex formation under such conditions was confirmed by His-DHX15 binding to CMTr1 followed by capturing the complexes on nickel-charged agarose beads .(h)RNA secondary structure, the minimum free energy (MFE), and the probability of individual base pairs were predicted using the ViennaRNA package , in part(i)http://fantom.gsc.riken.jp/5/suppl/Hon_et_al_2016/data/assembly/lv3_robust/. For further analyses, we considered only 52 nt 5′-terminal fragments of transcripts, which corresponded in length to the size of the 5′-terminal hairpin structure present in the substrates used in this work. As a result, 279 252 non-identical 52 nt sequences  were retained, which are hereafter referred to as the FANTOM dataset of 5′ ends (file FANTOM.bed.gz is available for download from ftp://genesilico.pl/iamb/data/CMTr1/).Sequences of human transcripts with accurately mapped 5′ ends, reported in , were obExperimentally determined secondary structure of the human transcriptome was extracted from the Structure Surfer database , which wThe KNIME data processing platform (v. 3.3.2)  and a se3.(a)To identify protein partners of human cap1 methyltransferase (CMTr1) that could regulate its activity, we performed immunoprecipitation experiments. FLAG-tagged CMTr1 was expressed in HEK293 cells (HEK293 Freestyle), and complexes were co-immunoprecipitated from the cell extracts with the use of anti-FLAG antibodies. Among all proteins co-immunoprecipitated with CMTr1, the highest ranked, according to Mascot score, was a 95 kDa protein, identified by MS as the ATP-dependent RNA helicase DHX15 . The enrichment of DHX15 with FLAG-tagged CMTr1 was also confirmed by western blot analysis, using antibodies recognizing DHX15 .d). The CMTr1–DHX15 interaction was further confirmed by a reciprocal Co-IP experiment using endogenous and FLAG-tagged DHX15 as bait . DHX15 co-immunoprecipitated endogenous CMTr1 and other G-patch-containing proteins previously known as DHX15 interactors . It has been shown previously that the OB-fold domain of DHX15 is responsible for interacting with G-patch-containing proteins [in vitro studies, except for G-patch, which we were not able to purify as a homogeneous peptide .To determine whether the G-patch domain of CMTr1 mediates the interaction with DHX15, we performed co-immunoprecipitation (Co-IP) experiments with the N-terminally truncated version of CMTr1 (CMTr1Δ135). Results of the MS identification and western blot analysis demonstrated that the G-patch domain of CMTr1 is responsible for the interaction with DHX15 d. The CM as bait b,c. DHX1proteins . However(b)−1 . Second, we developed two types of derivatives of the RNA64 substrate (a) to introduce a 23 bp double-stranded structure in their 5′ ends. First, RNA64 + 23 duplex comprised RNA64 and a 23 nt oligonucleotide fully complementary to the 5′ end of RNA64. According to RNAcofold, the dimeric RNA64 + 23 structure had predicted MFE of −47.80 kcal mol−1. Second, an RNA92 was constructed, where the 5′ end of RNA64 was extended by the addition of the aforementioned 23 nt sequence, connected by a GAAA tetranucleotide linker  linker. At the 5′ end of RNA92, we included an additional G residue to enable efficient transcription by a T7 RNA polymerase. The resulting RNA folded into a stem–loop structure with 23 canonical base pairs and a wobble (G○U) pair formed by the extra G residue. According to RNAfold, the RNA92 structure had a predicted MFE of −49.10 kcal mol−1. We have also developed a series of RNA92 variants, in which the 3′ end was shortened by 24 residues: RNA68 and its derivatives . The secondary structures of CMTr1 substrates  were verified by the SHAPE method . The experimentally derived SHAPE reactivity profiles were found to support the predicted secondary structures of our RNA substrates, and the structures presented in this work are obtained based on SHAPE-assisted predictions .Based on the observation that DHX15 forms a complex with CMTr1 , we hypoubstrate a to intrb).Compared with the original RNA64 substrate, which had a relatively weakly structured 5′ end, the methylation of the bi-molecular RNA64 + 23 substrate was reduced by approximately 30%. However, the methylation of the single-stranded RNA92 substrate containing a hairpin at the 5′ end was strongly (approx. 20-fold) diminished compared with the original RNA64 substrate. Likewise, an approximately 30-fold decreased cap1 methylation was observed for the RNA68 substrate, which was used in further experiments b.c; electronic supplementary material, figure S3). This effect is attributed to the 2′-O-ribose methyltransferase specificity of CMTr1 as shown in electronic supplementary material, figure S4. The stimulatory effect of DHX15 on CMTr1 activity was minimal for RNA64, which has a relatively weakly structured 5′ end, and it was very strong for RNA92 and RNA68 substrates, in which the 5′ end is highly structured, and in which the first transcribed nucleotide is involved in base-pairing (figure 2d). Thus, the formation of the CMTr1–DHX15 complex appears to be important for the effective methylation of substrates containing the base-paired 5′ regions.For comparison, we measured the methyltransferase activity of CMTr1 towards the aforementioned substrates in the presence of DHX15. DHX15 did not exhibit cap1 methyltransferase activity independently of CMTr1; however, upon addition of DHX15, CMTr1 methylated RNA more efficiently  responsible for the interaction. Similarly to the full-length CMTr1, CMTr1Δ135 methylated efficiently the RNA64 substrate , whereas its activity towards RNA68 was lower. However, in contrast to the activity of full-length CMTr1, DHX15 had negligible influence on methylation of RNA68 by CMTr1Δ135. This result strongly suggests that direct interaction of CMTr1 and DHX15, mediated by the G-patch domain of CMTr1, is required for the stimulation of CMTr1 methyltransferase activity on RNA with a highly structured 5′ end .Figure (c)a). Changes introduced in the RNA68 sequence ranged from single nucleotide substitutions that interfered with the ability of the very first 5′-terminal residues to form base pairs, to a series of substitutions that weakened the entire hairpin present in the original RNA68 substrate . The results of in vitro methylation assays show that RNAs with a relatively stable secondary structure at their 5′ ends were poorly methylated by CMTr1 alone (b). Importantly, while the liberation of the target residue (the first nucleotide of the transcript) from base-pairing has a relatively mild positive effect on methylation efficiency by CMTr1, the disruption of a long secondary structure has a strong effect. Thus, the presence of a relatively long helix in the 5′ end of the substrate has a strong inhibitory effect on CMTr1 activity, even if the target residue is unpaired. Upon addition of DHX15 to the methylation reaction, RNA68(v1–v4) substrates were methylated more efficiently . The effect of helicase activity of DHX15 was consistent with the strength of the secondary structure, which has to be unwound for the CMTr1 methylation to access the 5′-terminal residues in a single-stranded conformation. The regulatory effect of DHX15 on CMTr1 activity was stronger on RNA substrates with a long uninterrupted helix. However, it was also clearly seen on the RNA68v4 substrate, in which the series of canonical base pairs in the helix was interrupted by multiple mismatches.To further elucidate the influence of RNA secondary structure on the CMTr1 activity we prepared four derivatives of the RNA68 substrate that differ in the strength of secondary structure in the 5′-terminal region a. Changer1 alone b. Import(d)DHX15 helps CMTr1 in methylation of structured RNA substrates. Hence we wanted to check if the reciprocal effect takes place. We found that the ATPase activity of DHX15 was stimulated by CMTr1, but not by the CMTr1Δ135 variant , regardless of the presence of RNA (data not shown), which provides further evidence for direct interactions between these two proteins mediated by the G-patch domain of CMTr1. Firstly, we showed that DHX15 exhibited the strand displacement activity towards a bi-molecular double-stranded RNA (RNA40 + 23) and its variants, which had essentially the same sequence and structure as the single-stranded RNA68 variants, with the GAAA loop replaced by a blunt end . Secondly, we tested the influence of CMTr1 on the strand displacement activity of DHX15. These tests were carried out on the uncapped RNA40 + 23 duplex as well as on the RNA64 + 23 duplex, in which RNA64 was 5′-capped . In none of the cases, did we observe any increase in the strand displacement activity due to the addition of CMTr1 to DHX15 . Negligible strand displacement was observed due to CMTr1 alone, which explains its poor activity on RNAs with highly structured 5′ ends. Interestingly, we observed inhibition of strand displacement activity of DHX15 by CMTr1Δ135 on the capped RNA64 + 23 substrate and, to a lesser extent, on the uncapped RNA40 + 23 substrate. The inhibitory effect of CMTr1Δ135 may be due to its binding to the RNA substrate , while being unable to interact with DHX15, which may result in blocking DHX15 from exerting its strand displacement activity. This hypothesis requires further investigation including structural studies.(e)in vivo secondary structure determination, as deposited in the Structure Surfer database [in vivo secondary structure that can be an obstacle for CMTr1 activity but can be potentially unwound by DHX15.To assess the potential biological relevance of the identified functional interaction between CMTr1 and DHX15, we analysed the prevalence of secondary structure at 5′ ends of human RNAs that are potential substrates for CMTr1. We focused the study on 5′-terminal regions of human transcripts with accurately mapped 5′ ends  and seledatabase . Ultimat4.O-methylation on the first transcribed nucleotide. This additional methylation enhances translation of mRNA during oocyte maturation [In higher eukaryotes, 5′ ends of all mRNAs and many non-coding RNAs (including long non-coding RNAs (lncRNAs), small nucleolar RNAs (snoRNAs), and the majority of small nuclear RNAs (snRNAs)) are modified by ribose 2′-turation ,34 and fturation . Beyond turation .7GpppGAUC), thereby revealing the mechanism of CMTr1 interactions with the 5′ terminus of a capped RNA [Recently, we have determined the crystal structures of the active CMTr1 catalytic domain in complex with a methyl group donor SAM and a capped 4 nt ribonucleotide , and interactions of CMTr1 with the 3′-terminal part of the substrate (beyond the base-paired region). In our experiments, CMTr1 did not show any strand displacement activity on its own  and we saw no evidence for sequence specificity (data not shown). We suspect that the RNA structure plays the strongest role, including the possibility that the 3′-terminal part of the substrate may contribute to steric interference if the folding of the 5′-terminal part brings the rest of the molecule too close to CMTr1.In line with the potential requirement for help in removal of the secondary structure from the 5′ end of the RNA, we found that CMTr1 interacts strongly with an ATP-dependent RNA helicase DHX15. Thus far, DHX15 and its yeast homologue Prp43 have been implicated in diverse cellular functions involving RNA metabolism, including splicing and ribosome biogenesis, but not in RNA modification. It is known that DHX15/Prp43 distribution between individual target pathways is regulated by its interplay with the various G-patch protein cofactors . DHX15/PCMTr1 is a G-patch protein, and we found that it binds DHX15. Co-IP experiments showed that a CMTr1 variant that lacks the G-patch domain does not bind DHX15, and it also cannot be aided by DHX15 in the methylation of RNAs containing strongly structured 5′ termini. This provides evidence of G-patch-dependent interaction between CMTr1 and DHX15, which enables CMTr1 to methylate efficiently all types of capped substrates, regardless of their secondary structure. The mechanistic details of this process remain to be elucidated.Based on the available data, we speculate that CMTr1 and DHX15 form a relatively stable complex, using the G-patch domain of CMTr1 and the OB-fold domain of DHX15 , which can bind capped RNAs. The CMTr1 can either directly methylate the first transcribed nucleotide in RNAs with unstructured 5′ termini, or use the help of a physically associated DHX15 protein to free the 5′-terminal region from base-pairing, thereby making it available for methylation . We were unable to see a stimulatory effect of CMTr1 on the DHX15 strand displacement activity with any of the RNA substrates used. On the other hand, the decoupling of physical interactions between the enzymes by deletion of the G-patch domain of CMTr1 leads to inhibition of that activity. Additional studies, in particular, structure determination for the CMTr1–DHX15 complex with a substrate RNA and detailed biochemical analyses of kinetic parameters, are necessary to understand the interplay of these enzymes.RNA methylation aided by an RNA helicase has been reported in the course of rRNA modification mediated by snoRNAs during ribosome production in human cells . It was E. coli [E. coli 23S rRNA, namely m7G2069 and m2G2445. Both domains of RlmKL cannot bind helix 74 at the same time in the folded structure of the 23S rRNA, and the RlmL domain preferentially recognizes and methylates the single-stranded substrate rather than the duplex substrate, suggesting that helix 74 should be unwound to serve as a substrate for m2G2445 methylation. In this case, an RNA unwinding activity was found to be associated directly with RlmKL and in vitro assays have not indicated any requirement for the additional helicase activity [The requirement for RNA unwinding activity to enable methylation of highly structured substrates has also been reported for RlmKL methyltransferase in E. coli . This enWhile this work was under review, another study reported CMTr1–DHX15 interactions . The aut6A alters local RNA structure and acts as a dynamic switch that can control the RNA-structure-dependent accessibility of RNA binding motifs [O-Methylation was also suggested to affect RNA folding and structural stability [Thus far, RNA methylation has been found to influence RNA structure. In particular, mg motifs ,47. 2′-Otability . Here, w


Although porcupine quill injuries are common in dogs, the detailed appearance of the quill on diagnostic ultrasound, computed tomography, and magnetic resonance imaging has not been sufficiently described. A 4-year-old, intact, female Jack Russel terrier presented with severe neck pain and ataxia after an altercation with a porcupine 2 weeks earlier. Radiology, diagnostic ultrasound, computed tomography and magnetic resonance imaging were all utilised to identify a quill imbedded in the cervical vertebral canal and cervical musculature and were compared to each other. Surgical removal of the quill, guided by imaging findings, led to the resolution of the clinical signs in the patient. Previous ultrasound imaging reports have just stated that the quill consists of paralell hyperechoic lines, and do not mention the finer hyperechoic lines inbetween and do not try to provide a reason for the appearance. Previous computed tomography (CT) reports just mention identifying the quill on CT images (whether or not CT could identify the fragments), but do not go into detail about the attenuating appearance of the quill nor try to relate this to the composition of the quill. This is to the authors’ knowledge the first report with detailed imaging descriptions of a case of cranial cervical vertebral canal porcupine quill foreign body in a dog. This is also the first report to allude to a possible difference in imaging findings related to quill structure because of keratin orientation and melanin content. The ideal imaging modality to use remains elusive, but ultrasound, computed tomography and magnetic resonance imaging could all identify the quill. Hystrix africaeaustralis, are not unusual in southern Africa. Quill migrations are common, but migration into the central nervous system is rare (Daoust Erethizon spp.) or ridged or scaled (African or Hystrix spp.), which aids migration as well as being an irritant, and their often contaminated nature  at 18.3 mL/h, an intravenous constant rate infusion of fentanyl  at 3 μg/kg, and diazepam  intravenously at 0.5 mg/kg every 8 h. Gabapentin  and carprofen  were added because pain control was not adequate initially . The diazepam was concurrently decreased to 0.2 mg/kg intravenously every 4 h.Orthogonal cervical radiographs were within normal limits. Soft tissue ultrasound was performed with a 10-MHz linear array transducer , over the dorsal cranial cervical region, and revealed what appeared to be two porcupine quill fragments , which iAn MRI study in dorsal recumbency was performed the following day , to visualise the cranial cervical spinal cord and surrounding tissues. Only pre-contrast images were obtained as the administration of contrast at that time was not deemed necessary .Musculus rectus capitus ventralis, from its origin on the skull caudally, had an incomplete sagittal T2W hyperintense tract within it, with hyperintensity of the rest of the muscle adjacent to the tract. At the level of the atlanto-occipital joint, there was a T2W triangular defect in the muscle that was continuous with the joint space dorsally; thus, penetration and synovitis of the joint were suspected  and T1W hypointense (similar to the cortices of the vertebrae), single 66 mm long × 4.6 mm wide tubular structure, with a 1-mm hyperintense central core (isointense to muscle)  and e. Tuspected . This deuspected . The cauuspected  and f. TM. rectus capitus ventralis, as noted on MRI, were not seen initially, but on retrospective viewing a poorly defined region of subtle irregular contrast enhancement (HU 95) was noted with a triangular less attenuating focus, believed to be the same presumed tract seen on MRI. Overall, the CT and MRI findings correlated well. However, with CT, less evidence for a migratory tract was identified, and spinal cord changes were greatly underestimated. Computed tomography was highly suggestive of two fragments, which were not noted on MRI, with the caudal migration of the quill suspected.Pre- and post-contrast helical CT  was performed the next day, immediately pre-operatively, to compare the findings with that of MRI. A soft tissue window (window width of 350 Hounsfield units [HU] and window level of 40 HU) was used with 2-mm slice thickness and 50% reconstruction increments. For the post-contrast scan, iohexol  was administered intravenously at 1.5 mL/kg and the scan commenced 2 min after the end of injection. A linear mildly hyperattenuating (HU of 115–160) quill was seen extending dorso-caudally in the same orientation as seen on the MRI . HoweverM. rectus capitus, M. spinalis et semispinalis cervicis and Mm. multifidus were incised to expose the right side of the spinous processes. Within the abovementioned musculature, a pocket of fluid was identified with the 60-mm long quill fragment embedded in a caudo-dorsal direction. The quill was removed. A second, shorter fragment, 25 mm long, was identified entering the spinal canal at the level of the C1–C2 inter-arcuate space and was surgically removed after a partial dorsal laminectomy was performed (A dorsal midline incision was made that extended from the external occipital protuberance to the level of C4, exposing the nuchal ligament. The right dorsolateral cervical muscles were separated after an incision through the median fibrous raphe, and the right erformed . This frThe patient improved daily during 4 days’ post-operative hospitalisation and at discharge on the 4th day demonstrated mild ataxia even though neck guarding was still present. The patient had good neck mobility in all directions.This is a retrospective case report describing a client-owned dog using data retrieved and reviewed from clinical records of our academic hospital. The dog, during its period of hospitalisation, was treated and housed according to standard hospital protocols for the management of client-owned pets. No tests or treatments were conducted for research purposes. The owner provided standard signed consent for the data and images to be used for publication.The route by which migrating foreign bodies penetrate the central nervous system is often unclear (Dennis et al. Radiographs are of little benefit in identifying foreign bodies such as plant material and wood or porcupine quills (Grahn et al. Hystrix spp. have solid longitudinal keratin ‘stiffeners’ within the quill core that converge from the cortex towards the centre (Vincent & Owers Ultrasound findings in our report correlate well with literature reports (Grahn et al. The use of MRI has only been reported twice in the diagnosis of a quill foreign body in a dog (Flesher, Lam & Donovan The use of CT clearly demonstrated the quill in this case and was in the authors’ opinion superior to demonstrate the quill, contradictory to some of the literature. A case of an intra-cranial porcupine quill described the HU as ranging from 52 to 68 (Sauvé et al. Quills are reported to contain air Rose , but theHystrix spp. quills are longer and also of larger diameter than those of Erethizon spp. (Yang et al. It is unclear as to why the quill in our case was so easily and completely identified compared with previous reports, but because This is, to the authors’ knowledge, the first report with detailed diagnostic imaging descriptions in a case of a porcupine quill foreign body in a dog. This is also the first report to allude to a possible difference in imaging findings related to quill structure because of keratin orientation and melanin content. The ideal imaging modality to use remains elusive, but US, CT and MRI could all identify the quill. However, diagnostic ultrasound should be the first imaging modality used because of being readily available in practice and low cost.