Receptor-transporting protein 1S (RTP1S) is an accessory protein that mediates the transport of mammalian odorant receptors (ORs) into the plasma membrane. Although most ORs fail to localize to the cell surface when expressed alone in nonolfactory cells, functional expression of ORs is achieved with the coexpression of RTP1S. However, the mechanism for RTP1S-mediated OR trafficking remains unclear. In this study, we attempted to reveal the mode of action and critical residues of RTP1S in OR trafficking. Experiments using N-terminal truncation and Ala substitution mutants of RTP1S demonstrated that four N-terminal amino acids have essential roles in OR trafficking. Additionally, using recombinant proteins and split luciferase assays in mammalian cells, we provided evidence for the dimer formation of RTP1S. Furthermore, we determined that the 2nd Cys residue is required for the efficient dimerization of RTP1S. Altogether, these findings provide insights into the mechanism for plasma membrane transport of ORs by RTP1S. Olfaction is mediated by olfactory receptors (ORs)Many ORs fail to be transported into the plasma membrane and remain in the endoplasmic reticulum when they are expressed heterologously in nonolfactory cells , 9. Rece10–14–The amino acid sequences of RTP family proteins are highly conserved. The RTP family consists of type II transmembrane proteins with a cytoplasmic N terminus and a transmembrane domain (TM domain) close to the C terminus. The TM domain and the outer cellular region of RTP1S seem to play a role as the signal promoting OR localization in a lipid raft on the cell membrane . AlthougIn this study, we examined the role of the N-terminal amino acids in OR traffic activity. We also performed expression, purification, and structural characterization of the intracellular domain of RTP1S. The recombinant intracellular domain of RTP1S formed a dimer. We also provided evidence for the dimer formation of RTP1S in mammalian cells and demonstrated that the 2nd Cys residue is required for the efficient dimerization of RTP1S.A previous study showed that the truncation of nine N-terminal amino acids significantly impaired the OR traffic activity of RTP1S . To idenA and Figs. 1B and Fig. 1C and Fig. S1). Thus, the 21 amino acids from the 177th to the 198th amino acid of RTP1S before the TM domain seem to have no significant role in OR transportation into the plasma membrane.Cell-surface expression of ORs with the coexpression of RTP1S truncation mutants was analyzed by FACS analysis. Truncation up to Ser-4 (RTP1SΔN4) weakened the OR trafficking activity of RTP1S A and 2. D and A–C). Olfr1484 was unable to respond against acetophenone in the presence of RTP1S_S4A. We observed linear correlations with significantly high coefficients (R2 = 0.78–0.99) (D–F).To further evaluate the role of each amino acid residue at the N terminus of RTP1S in OR trafficking, we created Ala replacement mutants and coexpressed them with three different ORs D and 4. 78–0.99) , D–F.Fig. S2). RTP1SΔN2, RTP1SΔN4, RTP1S_C2A, and RTP1S_S4A showed relatively weak binding to the ORs compared with WT RTP1S .We also checked the protein expression level of all N-terminal truncation or Ala replacement mutants. Essentially, the expression of all mutants was not affected . In size-exclusion chromatography–multiangle laser light-scattering (SEC-MALS), the purified RTP1S_C2-Strep appeared as two peaks, the major one with a molecular mass of 43.3 kDa and the minor one with a molecular mass of 21.8 kDa (B). Given that the estimated molecular mass of RTP1S_C2-Strep was 22.0 kDa, these are likely to correspond to dimer and monomer forms, respectively. We also expressed and purified another TM deletion mutant, RTP1S_C3, with a Strep-tag at the C terminus (RTP1S_C3-Strep). The recombinant RTP1S_C3-Strep seemed to form larger oligomers .To study the detailed mechanism of RTP1S, we tried to express and purify the TM domain deletion mutant RTP1S_C2. RTP1S_C2, with a Strep-tag at the C terminus (RTP1S_C2-Strep), was then expressed in graphies A. In siz21.8 kDa B. Given C). The effect changed with the incubation time and the concentration of DTT. It is plausible that the dimer formation of RTP1S_C2-Strep is mediated by disulfide bonds. The peak for the oligomeric RTP1S_C3-Strep also decreased with the addition of DTT, and the elution time of that peak was delayed with the increase in DTT concentration . Thus, disulfide bonds are also responsible for the multimerization of RTP1S_C3-Strep.Incubation with dithiothreitol (DTT) induced a decrease in the dimer with an increase in the monomer C. The efC), suggested that Cys-2 plays a critical role in the homodimerization of RTP1S.We also expressed and purified RTP1S_C2-Strep mutants with the truncation of the N-terminal amino acids. Size-exclusion chromatography analyses on HPLC showed that the dimer formation of RTP1S_C2-Strep disappeared only after deleting the 2nd amino acid, Cys-2 (RTP1S_C2ΔN2-Strep) . These rFig. 7FACS analysis showed that the cell-surface expression levels of three ORs by the RTP1S mutant that lacks Cys-2 (RTP1SΔN2) decreased by 54–67% , and theA). The protein expression levels of all four RTP1S variants were almost the same as that of RTP1S when they were expressed by the pCI vector . Among them, only RTP1S fused with Lg at the N terminus (Lg-RTP1S) had lost OR trafficking ability .We examined RTP1S dimer formation in mammalian cells by a binary split-luciferase complementation method using NanoLuc Technology . In thisFig. 9B). However, complementation of Nanoluc was not observed in the combinations of the RTP1S variants across the membrane (Sm-RTP1S/RTP1S-Lg) (B). These results indicated that RTP1S proteins interact with one another, consistent with the idea that it forms a homodimer in mammalian cells.The RTP1S fusion variant genes were inserted into the pBit vector to make the expression level suitable for the assay. All combinations of RTP1S fusion variants were coexpressed in HEK293T cells. Among them, significantly high bioluminescence associated with complementation of split Nanoluc was observed when RTP1S-Lg and RTP1S-Sm were coexpressed B. HoweveTP1S-Lg) B. These B), we examined whether RTP1SΔN2 shows a homotypic interaction. The bioluminescence of the cells expressing RTP1SΔN2 fused with Lg and Sm at the C terminus was significantly lower than that of RTP1S (C). However, RTP1SΔN2 still exhibited bioluminescence, which means that it has a homotypic interaction at the C terminus. The FKBP–Sm fusion protein as a control did not show a nonspecific interaction with RTP1S–Lg . From these results, it was presumed that there are other parts involved in the dimer formation of RTP1S. The 2nd Cys residue located in the cytosolic domain seemed to be required for efficient dimerization of RTP1S.Because the purified RTP1S_C2ΔN2 did not form the homodimer B, we exaof RTP1S C. HoweveD). The results showed homotypic interactions of RTP2 and RTP4, although the efficiencies seem to be lower than that of RTP1S. Then we examined whether RTP proteins can form heteromers. Our data show that RTP proteins form heteromers with each other (E). These results suggested that homo- and heterotypic dimerization of RTP is a property common to RTP family proteins.Next, we examined whether other RTP family proteins, RTP2 and RTP4, also show homotypic interactions. RTP2 or RTP4 fused with Lg and Sm at their C termini were coexpressed, and the bioluminescence was measured D. The rech other E. These In the olfactory sensory neuron cells, RTP1S and RTP2 are expressed and transport ORs into the plasma membrane . TherefoFig. S6), supporting the idea that Cys-2 plays a conserved role in RTP1S function in mammals.In the eight N-terminal amino acids of RTP1S, six residues, including Cys-2, are conserved between various mammalian species (A). Truncation of the four N-terminal amino acids of RTP1S_C2 (RTP1S_C2ΔN4) caused a loss in traffic activity for Olfr599 (B–D). The two amino acid replacements in RTP1S (C2S/G8C) significantly impaired OR-transporting activity (43.6% against RTP1S) (E–G). In contrast, the RTP2 mutant with the two amino acid replacements (S2C/C8G) exhibited enhanced activity (157% against RTP2) (E–G).RTP1S and RTP2 share 74% 163/221) identity and 81% (180/221) similarity in the mouse. The OR trafficking ability of RTP2 seems to be lower than that of RTP1S . Four amFig. 10 Olfr599  (21). Th identityTogether, these data suggest that Cys-2 and Ser-4 in the N terminus of RTP1S are especially important for efficient trafficking of ORs. However, the minimum elements of RTP1S required for OR transport into the cell membrane differ depending on the OR of the transport partner.In this study, we conducted functional and structural analyses of RTP1S to unveil the mechanism of OR transport to the plasma membrane. We showed that RTP1S forms a homodimer and that specific residues located at the N terminus play essential roles in trafficking ORs.This is the first report on the homodimer formation of RTP1S evidenced by both recombinant protein analysis and living cell assays. The 2nd cysteine deletion or replacement decreased the levels of RTP1S dimerization and decreased the level of ORs localized on the cell surface , and 8 b24–A previous study with RTP1 and RTP2 double-knockout mice showed that some ORs could independently localize and be functional in the cell membrane during OSN maturation without RTP1 and RTP2 , 34, 35.Evaluation of the functional activity of recombinant RTP1S is difficult because we used indirect methods such as OR trafficking or responsiveness. Although it is thought that ORs become structurally stable by interacting with RTP1S during membrane transport, the structure of RTP1S itself may also be stabilized by the interaction with ORs.i.e. X-ray crystallography, NMR, or cryo-EM) of RTP proteins would allow us to conduct various approaches, including protein–protein docking and molecular dynamics simulation, for investigating the interaction between RTP and ORs using the 3D theoretical model of ORs. Further analysis is necessary to unveil the membrane transport mechanism of OR by RTP1S to understand how RTPs transport hundreds of ORs from the endoplamsic reticulum to the plasma membrane.For further studies, high-resolution structural analyses (Rtp1S variants with the Strep-tag sequence (WSHPQFEK) at the C terminus were subcloned into the pET23b vector. They were expressed in E. coli BL21 star (DE3) cells. Purification of RTP1S_C2-Strep and RTP1S_C3-Strep was carried out by several steps of chromatography using StrepTactin–Sepharose high performance , Resource Q , and a gel-filtration column Superdex 75 . Purified RTP1S_C2-Strep and RTP1S_C3-Strep in 20 mm phosphate buffer, pH 8.0, with 100 mm NaCl were concentrated by ultrafiltration and used for analysis immediately.All DNAs for m phosphate buffer, pH 8.0, 100 mm NaCl). Measurement of molecular mass was carried out under the same conditions by SEC-MALS using a multiangle light-scattering detector  and a differential refractive index detector .The purified RTP1S_C2-strep and RTP1S_C3-Strep were analyzed using an HPLC system with OH-Pak SB-804HQ . An aliquot (100 μl) of the RTP1S solution (0.3 mg/ml) was injected onto the column with buffer  were maintained in minimum essential medium Eagle's  containing 10% fetal bovine serum  and 0.5% antibiotic/antimycotic mixed stock solution  at 37 °C in 5% COd-lysine–coated 96-well plates (Thermo Fisher Scientific). Plasmid DNAs were transfected using Lipofectamine 2000 (Invitrogen). Then, each Rtp1S was subcloned into the pBiT1.1-C (TK/LgBiT), pBiT2.1-C (TK/SmBiT), pBiT1.1-N (TK/LgBiT), and pBiT2.1-N (TK/SmBiT) vectors. Combinations of plasmids for RTP1S fusion variants were transfected into HEK293T cells. After 24 h post-transfection, the medium was replaced with 40 μl of 10 mm HEPES, and we followed the manufacturer's protocols for measuring luciferase activities. Luminescence was measured using a GloMax-Multi Detection System (Promega).We used the Nano-Glo Live Cell Assay System  for the split luciferase reconstruction assay. HEK293T cells were plated on poly-Olfr1484, Olfr1377, and Olfr599 were subcloned downstream of the Rho-tag DNA fragment. The Rtp1S gene was inserted into the pCI expression vector without the Rho-tag (pCI-RTP1S). Through PCR-based mutagenesis on pCI-RTP1S, plasmids for RTP1S mutants were constructed. The sequences of all plasmids were verified by sequencing.The DNA fragment for the Rho-tag (MNGTEGPNFYVPFSNATGVVR) was subcloned into the pCI mammalian expression vector (Promega). Then, the genes d-lysine–coated 96-well plates (Thermo Fisher Scientific). Plasmid DNAs of ORs and RTP1S were transfected using Lipofectamine 2000 (Invitrogen). In addition, two luciferase constructs were used, including a firefly luciferase gene driven by a cAMP-response element (CRE-Luc) and a Renilla luciferase gene driven by a constitutively active SV40 promoter (pRL-SV40) that was used as an internal control for cell viability and transfection efficiency. For each 96-well plate, 10 ng of CRE-Luc, 5 ng of pRL-SV40, 5 ng of OR, and 5 ng of RTP1S were transfected. Twenty four hours post-transfection, the medium was replaced with 25 μl of odorant solution diluted in CD293 (Invitrogen) and incubated for 3.5 h at 37 °C in 5% CO2. We followed the manufacturer's protocols for measuring firefly luciferase (Luc) and Renilla luciferase (RL) activities. Luminescence was measured using a GloMax-Multidetection System (Promega). The relative response of ORs was calculated as (Luc/RL − Luc/RLmin)/(Luc/RLmax − Luc/RLmin), where Luc/RLN represents the mean value from the replicate wells of a certain sample, and Luc/RLmin represents the mean value from the minimal response in the experiment.The Dual-Glo luciferase assay system (Promega) was used for the luciferase assay . HEK293Tm NaN3). Then, the cells were transferred to a tube for incubation with the anti-rhodopsin 4D2 antibody  for 45 min on ice and then with phycoerythrin (PE)-conjugated donkey anti-mouse IgG  for 30 min on ice. Each antibody was diluted in staining buffer. To stain dead cells, 7-amino-actinomycin D (Merck Millipore) was added before analysis. The cells were analyzed using a BD FACS Canto II flow cytometer  and a BD LSRFortessa cell analyzer (BD Biosciences) with gating allowing for 10,000 GFP-positive, single, spherical, and viable cells, and the measured PE intensities were analyzed and visualized using FlowJo  and washed with staining buffer (PBS containing 2% (v/v) fetal bovine serum and 15 mg FlowJo .m Tris, pH 8.0, 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1× complete protease inhibitor mixture (Merck Millipore)). The lysates were incubated with Dynabeads (Thermo Fisher Scientific) conjugated with the anti-Rho4D2 antibody (Merck Millipore) for 10 min at room temperature and washed with PBS buffer, pH 7.4, containing 0.02% Tween 20. The bound proteins were eluted by incubation with 1× SDS sample buffer at room temperature for 2 h and at −80 °C overnight and then subjected to Western blotting.HEK293T cells in 35-mm dishes were transfected with the plasmids to express the N-terminally Rho-tagged OR and RTP1S variants and/or the negative control. Twenty four hours after transfection, cells were lysed with RIPA buffer . The membranes were blocked and incubated with primary antibodies (anti-Rho4D2 antibody or anti-RTP1 antibody (Invitrogen)) followed by horseradish peroxidase-conjugated secondary antibodies  using iBind Western Systems (Thermo Fisher Scientific). The signals were detected using Western BLoT Quant HRP substrate  using the C-DiGit System  according to the manufacturer's instructions.Amino acid sequences of RTP1S from various animals were chosen from the NCBI database. The animal species and accession numbers we used were as follows: mouse, ABU23737.1; human, AAT70680.1; rat, NP_001099340.1; dog, XP_025307127.1; gorilla, XP_004038226.1; flying fox, XP_006925887.1; Chinese hamster, XP_007606938.1; macaque, NP_001270896.1; horse, XP_001498142.1; cow, NP_001179185.1; star-nosed mole, XP_004675139.1; opossum, XP_001366241.1; koala, XP_020844727.1; and platypus, XP_001512958.1. The phylogenic tree and the amino acid alignment were analyzed using the ClustalW program.t test was performed using the built-in function in Microsoft Excel. The average is shown as the mean ± S.E.Multiple comparisons were performed using one-way or two-way analysis of variance using GraphPad Prism . Student's Y. F. and M. Y. conceptualization; Y. F., R. T., R. I., T. K., S. S., and K. I. data curation; Y. F. and R. T. formal analysis; Y. F., H. M., and M. Y. supervision; Y. F. and M. Y. funding acquisition; Y. F., R. T., R. I., and K. I. investigation; Y. F., I. O., H. M., and M. Y. methodology; Y. F. writing-original draft; Y. F. and M. Y. project administration; Y. F., H. M., and M. Y. writing-review and editing; R. T. validation; I. O. resources.


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Phylogeographic analyses based on NS5A domain 1 sequences indicated circulation of both locally and nationally epidemic strains. Relatively high frequency of Y93H (14.1%) was only detected in GT1b but not in other subtypes. High frequency of L31M was found in both GT2a (95.6%) and GT3b (98.7%) sequences. Due to the overlapping incidence of A30K, 96% of GT3b isolates had NS5A RASs combination A30K + L31M, which confers high levels of resistance to most NS5A inhibitors. No RASs were detected in GT6a strains. Meanwhile, baseline NS5A RASs fingerprints were also evaluated in 185 DAA treatment-naive GT1b patients with next generation sequencing method. Patients presenting with Y93H had statistically higher entropy of HCV NS5A sequences. Taken together, subtype-specific distribution patterns of NS5A RASs were observed. GT1b patients with higher HCV complexity tend to have a greater chance of Y93H presence, while GT3b patients are naturally resistant to current NS5A inhibitors and their treatment may pose a challenge to real-world DAA application.Resistance associated substitutions (RASs) can reduce the efficacy of direct-acting antiviral agents (DAAs) targeting hepatitis C virus (HCV) and lead to treatment failure. Clinical data of HCV NS5A RASs prevalence are limited in China and need to be investigated. A total of 878 unique patient samples with different genotypes (GT) (1b:  Hepatitis C virus infection is a global public health problem. HCV chronically infects about 3% of the world’s population, many of whom will develop liver cirrhosis and hepatocellular carcinoma .Hepatitis C virus has a high degree of genetic heterogeneity. It can be classified into seven GTs and numerous subtypes . In ChinDirect-acting antiviral agents, targeting primarily the HCV NS3/4A protease, NS5A protein or NS5B polymerase, have replaced pegylated-interferon based therapy due to generally high sustained virological response (SVR) rate and good tolerance . Among tin-vitro resistance to potent NS5B inhibitor sofosbuvir, was rarely seen at baseline and has been observed only in few patients at treatment failure . Roughly 1/8 of total samples ordered for HCV genotyping tests were randomly selected out for NS5A RASs determination. A total of 878 unique serum samples were included in the study. Each sample was given a de-identified code. The corresponding gender and age data were recorded. HCV genotyping and subtyping were performed as described previously .Another set of DAA-naïve serum samples were collected from 185 GT1b-infected chronic hepatitis C patients between April 2015 and September 2017 at Department of Infectious Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (hereinafter referred to as Shanghai Ruijin). Written informed consent was obtained from all patients before the study. HCV subtype was confirmed by RT-PCR and sequencing of the HCV core region as described previously . No patiAF009606) and the purified DNA product was sequenced by direct Sanger sequencing. Briefly, total viral RNA was extracted from 140 μL of serum with TIANamp Virus DNA/RNA Kit  according to manufacturer’s protocol. RNA was eluted with 50 μL of nuclease-free water and kept at −80°C for future use. HCV RNA was reverse transcribed and amplified for the first round with HiScript® II One Step RT-PCR kit  and 2 × phantaTM Master Mix  for the second round following the manufacturer’s instructions. PCR conditions included a predenaturation step at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s. A final 5-minute extension step at 72°C was added. PCR reaction contained 25 μL of 2 × reaction premix, 1 μL of each primer (10 μM), 5 μL of cDNA template, and nuclease-free water to a final volume of 50 μL. The first round PCR product was used as the template in the second round of PCR, which was done with the same reaction volume and conditions as the first round. Sanger sequencing of PCR products from both directions was performed using an automatic sequencer . Sequencing primers were the second round PCR primers. Chromatograms were visually inspected and analyzed using Sequencher . Mixed-calls were assigned where minority alleles composed >20% of chromatogram.For samples collected at Kingmed, NS5A region was amplified by nested primers  spanningAJ238799). The technical error rate was estimated by amplifying and sequencing Con1 plasmid exactly the same as the clinical isolates. A cut-off value (1%) was set as the minimum threshold in calling the presence of polymorphism.For samples collected at Shanghai Ruijin, HCV NS5A domain 1 sequence was determined by both Sanger sequencing and Next Generation Sequencing (NGS) as follows. HCV RNA was reversed transcribed and NS5A region was amplified as described previously except with the following modifications: The PCR reaction was catalyzed with Q5 High-Fidelity DNA Polymerase (New England Biolabs). For NGS multiplex sequencing, PCR products from the first round PCR reaction were amplified in the second round with nested primers which had sample-specific 7-bp barcodes additionally incorporated. PCR amplicons were purified with Agencourt AMPure Beads  and quantified using the PicoGreen dsDNA Assay Kit . After the individual quantification step, amplicons were pooled in equal amounts, and pair-end 2 × 300 bp sequencing was performed using the Illumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd . For the calling of nucleotide mutations and amino acid substitutions, each amplicon was compared to the reference sequence of HCV 1b-Con1 , 2a-JCH-1 (AB047640), 3a-PR87 (HQ912953), 3b-HCV-Tr (D49374) and 6a-HK33 (AY859526) retrieved from GenBank. HCV NS5A 1–309 nucleotides were aligned with reference sequences. GT3a and GT3b sequences were additionally aligned with reference sequences with available information of isolation regions, including countries from Europe , Asia , North America (United States), South America (Brazil) and Oceania , with details provided in the respective figure legends. The evolutionary history was inferred by using the maximum likelihood (ML) method based on the i (pi×ln pi)]/ln N, in which pi is the proportion of each different sequence, and N is the total number , and continuous variables were described as mean ± standard error (SEM). Statistical analyses were performed using GraphPad Prism software. Comparisons among multiple groups or between two groups were performed using Chi-square test or n = 489), GT2a (n = 203), GT3a (n = 60), GT3b (n = 78) and GT6a (n = 48). Samples of GT1a (n = 1) and GT6n (n = 7) were excluded from the study due to the limited sample size. No samples of mixed-subtype HCV infection were detected. The general information of these patients is summarized in The present study included 878 randomly selected unique patient samples collected at KingMed Laboratory from January to June 2018. The samples were taken from 27 provinces/municipalities . HCV genotyping was initially accomplished by TaqMan PCR and further validated by core-envelope 1 sequencing . Samplesn = 134, East n = 162, South n = 111, West n = 82), 203 GT2a sequences , 60 GT3a sequences , 78 GT3b sequences , 48 GT6a sequences  and five representative reference sequences . A circular ML phylogenetic tree is shown in Phylogenetic analyses of all NS5A sequences amplified were conducted to investigate the genetic relationship among them. After aligning with HCV strains of standard GTs, a 309-bp fragment of the partial NS5A region (H77 positions: 6258–6566 nucleotides) was utilized for HCV phylogenetic analyses. The phylogenetic tree included 489 GT1b sequences . Clinically relevant NS5A RASs were determined according to literatures . The majIn GT1b sequences, the proportion of Y93H was 14.1%. The residues of Q24, L28, L31, P32, S38, and A92 were highly conserved. Residues R30 and P58 were more polymorphic: 84 (17.2%) or 34 (7%) of 489 sequences harboring substitutions Q30 or S58, respectively.In GT2a sequences, L31M was found in 95.6% of GT2a subjects, indicating a homogeneously high frequency of RAS at this amino acid. No RAS was detected at Y93 position. The proportion of T24S, F28L, K30R, P58S, and C92S was as low as 2.5, 2.5, 1.0, 1.5, and 3.0%, respectively.Interestingly, totally different distribution patterns of RASs were noted in GT3a and GT3b isolates. Among the 60 GT3a sequences, only 3.3% were detected with Y93H and no other RASs were found. In contrast, 97.4 and 98.7% of GT3b sequences were detected with A30K and L31M mutation, respectively. Y93H was not observed in any GT3b sequences.As for GT6a sequences, substitutions of Q24K/R (18.8%), L28F (20.8%) and T58S (6.3%) were detected, but no Y93H was found.Coincidence of multiple RASs may further decrease the genetic barrier to resistance and toughen the treatment selection . We analp = 0.0083).Among the RASs listed, Y93H is primarily associated with reduced NS5A-targeting DAAs efficacy across all subtypes . Y93H waTo rule out the sampling bias at Kingmed Laboratory and further verify the Y93H prevalence in GT1b patients, a validation set of 185 GT1b-infected DAA-naïve patients samples collected at Ruijin hospital was tested for RASs frequency . The NS5p = 0.037) (The quasispecies genetic diversity between GT1a and GT1b was hypothesized to contribute to a higher level of DAA resistance in GT1a patients . To eluc= 0.037) , which sHepatitis C is an important cause of liver-related death. With the emergence of DAAs that have overwhelmingly transformed HCV treatment, WHO advocated the vision to eliminate the disease by 2030. However, clinical application of DAAs is highly concentrated in Europe and United States, but still rare in resource-limited countries and regions. Patients in China have less choice than those in United States or Europe, since only daclatasvir, asunaprevir, sofosbuvir, simeprevir, danoprevir, elbasvir/grazoprevir, ombitasvir/paritaprevir/ritonavir + dasabuvir (3D), sofosbuvir/ledipasvir and sofosbuvir/velpatasvir have currently been approved in mainland China for HCV treatment with different indications. For example, danoprevir can merely be applied with pegylated-interferon/ribavirin and to non-cirrhotic GT1b patients. Elbasvir/grazoprevir is suitable for GT1 and GT4 patients, while daclatasvir/asunaprevir and 3D regimen can only be applied to GT1 patients. Considering the high prices of these DAAs, reasonable and individualized treatment plans are demanded.Benefiting from the nationwide network of sample collection by Kingmed Laboratory, we randomly selected 878 samples with different subtypes from 27 provinces/municipalities across China. Different GTs have distinct geographic and demographic distribution patterns, which are consistent with a previously published large sample size survey . In geneNatural occurring NS5A RAS distribution was our initial study aim. However, due to missing information of DAA experience in Kingmed sampling procedures, we can not rule out the possibility that some of the samples may be from DAA-experienced patients. In our estimation, the ratio of DAA-experienced patients should be very low based on the following reasons. Firstly, original DAAs were not approved until the middle of 2017. For example, daclatasvir + asunaprevir was the first DAA regimen approved in mainland China in June 2017. Simeprevir, 3D regimen and sofosbuvir were then approved in China in the late 2017. Considering the treatment duration, samples collected in the first half year of 2018 would be mainly from DAA-naïve patients. Secondly, Kingmed Laboratory roughly has 14,000 samples ordered for HCV genotyping test yearly . We randp = 0.0083). To rule out biased RASs presence due to DAA experience in patients from Eastern China, which is the most populated and wealthy region and patients there may have higher chances of DAA exposure, we used 185 DAA-naïve GT1b patients samples collected at Shanghai Ruijin Hospital, harboring one of the biggest hepatitis C center located in Eastern China, to repeat the RASs study. We adopted both Sanger sequencing and deep sequencing to analyze RASs prevalence in this validation set. The detection limit of viral variants in quasispecies is usually about 10 to 25% for Sanger sequencing and 1% for NGS, respectively , but was highly resistant to daclatasvir, velpatasvir  and elbasvir  (GT3 HCV infections are among the most difficult to treat in DAA era . Our datin EC50) . The ovein EC50) . In our In conclusion, the prevalence of NS5A RASs can be frequent and diversified depending on HCV genotype/subtype and treatment regimen. Y93H in GT 1b, L31M in GT2a, A30K and L31M in GT3b NS5A sequences, contribute most to NS5A RASs prevalence in China. DAA-naïve GT1b patients with greater viral complexity had higher prevalence of NS5A 93H substitution. GT3b patients are naturally resistant to NS5A inhibitors. The information of baseline NS5A RASs may provide clinical treatment guidance. Due to the essential role of NS5A inhibitors, high cross resistance among different NS5A inhibitors, and long-term persistence of NS5A inhibitor-resistant HCV variants, patients who have baseline NS5A RASs should be cautious in their DAAs selection.The data collection and analysis for publication was approved by the Human Ethics Committee of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.JL, YF, LC, XG, XL, WT, and QX designed the research, analyzed results and drafted the manuscript. JL, YF, ZZ, XL, WC, HW, HZ, XG, and QX contributed to the clinical data collection and interpretation. All authors read and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Propionibacterium acnes (P. acnes) exerts immunomodulatory effects by increasing the phagocytic and tumoricidal activities of macrophages, promoting DC maturation, inducing pro-inflammatory cytokines production and increasing the humoral response to different antigens. Here, we evaluated the effect of P. acnes on a specific antitumor immune response elicited by a hybrid vaccine in a mouse melanoma model. Hybrid vaccine associated with P. acnes increased the absolute number of memory T cells, the IFN-γ secretion by these cells and the IgG-specific titers to B16F10 antigens, polarizing the immune response to a T helper 1 pattern. Furthermore, the addition of P. acnes to a hybrid vaccine increased the cytotoxic activity of splenocytes toward B16F10 in vitro and avoided late tumor progression in a pulmonary colonization model. These results revealed the adjuvant effect of a killed P. acnes suspension, as it improved specific humoral and cellular immune responses elicited by DC-tumor cell hybrid vaccines.Hybrid vaccines have been investigated in clinical and experimental studies once expresses total antigens of a tumor cell combined with the ability of a dendritic cell (DC) to stimulate immune responses. However, the response triggered by these vaccines is often weak, requiring the use of adjuvants to increase vaccine immunogenicity. Killed  Indeed, based on many clinical and experimental studies, vaccination with DCs pulsed with tumor lysate cells  thymidine (5μCi/mL) (Amersham Biosciences) for 24 hours at 37°C and 5% CO2. The cells were collected, washed and the cell number and viability were determined by counting cells stained with Trypan Blue (Gibco-15250061). 1x104 B16F10 cells were co-cultured with splenocytes from the vaccinated or control groups at a ratio of 1 target cell to 50 effector cells, to detect cytotoxicity toward melanoma (E). Spontaneous lysis (S) was determined by culturing B16F10 cells alone in complete medium. After a 3.5 hour incubation , the cells were harvested (PerkinElmer), and radioactivity was measured using a β-counter (MicroBeta2LumiJET-PerkinElmer). The percentage of lysed cells was determined by:B16F10 cells were cultured in complete medium containing [methyl-5 B16F10 cells seven days after the last immunization. Fourteen or twenty days later, the mice were euthanized, the lungs were extracted and the number of nodules was determined. When we could not count the lung nodules due to intense colonization, this parameter was plotted on the graphs as 500 nodules. None animal showed severe signs of illness following tumor formation or adverse outcomes.Animals were intravenously (iv.) challenged with 3x10The significance of the differences between the control and vaccinated groups was analyzed using one-way ANOVA followed by Tukey's Multiple Comparison Test using GraphPad Prism software version 5.0 . Differences were considered statistically significant at p<0.05.P. acnes to the hybrid vaccine increased the total IgG, IgG1, IgG2b and IgG2c titers specific to B16F10 antigens compared to immunization with the hybrid vaccine (p<0.05). As expected IgG2a was not detected in C57Bl/6 mice (data not show). Antibodies against melanoma antigens were not also detected in the PBS and P. acnes controls groups  were analyzed using ELISAs to detect the titers of IgG isotypes specific for B16F10 antigens. The mean IgG1 and IgG2c titers were calculated using the data from three independent experiments. The isotype ratio was obtained by dividing the mean IgG1 titer by the mean IgG2c titer.Cells were collected from the draining lymph nodes and the memory phenotype was evaluated . All trenaïve T cell numbers showed increased numbers of CD8 T cells in the Hybrid+P. acnes and P. acnes groups and an increased number of CD4 T cells in the P. acnes group only  and effector memory (EM) CD4 T cells compared to the Hybrid and PBS groups  (The Hybrid+ctively) .5±0.15) compared to the PBS (0.60x105±0.40) and Hybrid (1.05x105±0.05) groups  (5±0.10) CD4 T cells (P. acnes group were less than the numbers observed in the Hybrid+P. acnes group (Moreover the coadministration of the adjuvant and vaccine increased the absolute number of activated CD4 T cells (3.15x10) groups , but did) groups . The adm05±0.25)  and EM ( T cells  comparedes group .P. acnes to the hybrid vaccine increased the number of CM T cells (15.75x105±0.75) compared to the other groups   (P. acnes group (2.10x105±0.10) increased the absolute number of EM CD8 T compared with the Hybrid (1.05x105±0.05), PBS (0.25x105±0.05) and P. acnes (0.75x105±0.25) groups   and also05±0.10) , but no 05±0.10) . Moreove) groups .naïve CD4 T or CD8 T cells , 29.4±1.8% (PBS) and 26.3±1.9% (P. acnes) (Cells from the Hybrid+. acnes) .in vivo. Seven days after the second immunization, the animals were intravenously challenged with B16F10 cells, and the number of lung nodules was determined after 14  compared to the PBS and P. acnes groups (mean number of lung nodules in both group = 500) (P. acnes group displayed a reduced number of lung nodules (91.4 nodules) compared to the Hybrid (346.8 nodules), PBS (500 nodules) and P. acnes (500 nodules) groups   . However) groups . Thus, inodules) . Howevernization .P. acnes group, and the existing nodules were much smaller than the nodules observed in the other three groups  . In a meP. acnes was also revealed by the cytokine profiles of tumor-specific T cells stimulated in vitro with tumor antigens. Hybrid vaccine increased the production of IL-4 and IL-17 by CD4 T cells in relation to PBS group e did not alter their IFN-γ synthesis. However, P. acnes association to hybrid vaccine induced high levels of IFN-γ production and reduced IL-4 and IL-17 synthesis by CD4 T cells in relation to Hybrid group  described the presence of Th17 cells on tumoral microenvironment in mice and humans [et al. (2008) observed that Th17-polarized cells mediated destruction of advanced melanoma after being adoptively transferred to tumor-bearing mice [Kryczek d humans , but thed humans , 46. Neving mice . Moreoveing mice . Thus, fP. acnes improved Th1-specific immune response to a plasmid DNA T. cruzi vaccine reducing parasitemia compared to the control group [P. acnes potentiates or suppresses the Th2 response to OVA in a murine asthma model, depending on the P. acnes treatment protocol [P. acnes induces the activation of innate immune cells via TLR2 and TLR9 signaling [In our previous study, ol group . In addiprotocol , 27. Theignaling , 50 and ignaling , 39, 40.in vivo expansion of T cells after immunization with the hybrid vaccine, which increased the absolute numbers of total CD4 T and CD8 T cells compared to the PBS group  demonstrated that P. acnes association to a DNA vaccine containing 18 CD4+ T cell epitopes from human immunodeficiency virus (HIV) increased the proliferation of HIV-1-specific CD4+ and CD8+ T lymphocytes, including a higher percentage of CD4 T cells producing IFN-γ and TNF-α [Based on our results is not possible know if each T cell from Hybrid+nd TNF-α .in vitro. Splenocytes from the Hybrid+P. acnes group showed greater cytotoxic activity against B16F10 cells than the PBS, P. acnes and Hybrid groups  and NKT cells that also mediate tumor cell death. P. acnes has been shown to increase the tumoricidal activity of macrophages and NKT cells [P. acnes-treated mice are cytotoxic to melanoma cells, and one of the main mechanisms mediating the effect of this bacterium is nitric oxide release [Another mandatory immune response to eliminate tumor cells is the cytotoxic activity of immune cells; thus, we investigated this function in splenocytes from the treated groups d groups . This efKT cells , 18. We P. acnes was observed 20 days after challenge in vivo; however, tumor progression in the Hybrid was similar to the control group during the same period  demonstrated that intratumoral injection of P. acnes protected against skin melanoma progression in vivo and observed infiltrating T cells expressing TNF-α and IFN-γ at tumor lesions [Interestingly, the protective effect of the combination of the hybrid vaccine and e period . Moreoveid group . When mees group . It is p lesions .P. acnes to the hybrid vaccine on prolonging the inhibition of tumor growth, likely reflecting the increased production of T memory cells and their ability to kill tumor cells. The expansion of T memory cells was detected 24 hours after the second immunization with the hybrid vaccine and P. acnes, and this effect was maintained for 28 more days, when the last evaluation of tumor growth inhibition was conducted in vivo.Hybrid vaccines have been shown to prevent tumor growth , 36. NevP. acnes enhances the immunogenicity of the hybrid vaccine by improving the specific immune response elicited by this vaccine and prolonging its control of tumor growth. Moreover, this study presents an interesting approach that deserves further investigation for potential clinical applications.In summary, S1 Checklist(PDF)Click here for additional data file.S1 Fignaïve (CD44lowCD62Lhigh), EM (CD44highCD62Llow) and CM (CD44highCD62Lhigh) CD4 and CD8 T cells and degree of activation.Cells were gated in an FSCxSSC dot plot (A), and doublets were excluded (B). CD4 (C) or CD8 (D) T cells were selected and analyzed for the concomitant expression of CD62L and CD44 (E and F) or CD69 expression (G and H) to determine the percentages of (TIF)Click here for additional data file.S2 Fig+CD4+ and CD3+CD8+) (A) and subpopulations of naïve (CD44lowCD62Lhigh) (B), CM (CD44highCD62Lhigh) (C) and EM cells (CD44highCD62Llow) (D) are presented in the graphs. The percentage of activated CD4 and CD8 T cells (CD69+) (E) and their degree of activation based on CD69 mean fluorescence intensity (MFI) (F) were also investigated. ANOVA with Tukey’s post-test *p<0.05, **p<0.01.Seven days after the second vaccine dose, C57Bl/6 mice (n = 3) were challenge with B16F10 intravenously. Twenty days later, animals were euthanized, lung were extracted and from this tissue was obtained a cell suspension. Tumor infiltrating lymphocytes were enriched using Percoll gradient. Subsequently, the cells were stained with fluorochrome-conjugated monoclonal antibodies and analyzed using flow cytometry. The mean ± SEM percentage of CD4 and CD8 T cells (CD3(TIF)Click here for additional data file.


SCN1A gene, which encodes for the voltage-gated sodium channel NaV1.1, cause Dravet syndrome, a severe developmental and epileptic encephalopathy. Genetic testing of this gene is recommended early in life. However, predicting the outcome of de novo missense SCN1A mutations is difficult, since milder epileptic syndromes may also be associated. In this study, we correlated clinical severity with functional in vitro electrophysiological testing of channel activity and bioinformatics prediction of damaging mutational effects. Three patients, bearing the mutations p.Gly177Ala, p.Ser259Arg and p.Glu1923Arg, showed frequent intractable seizures that had started early in life, with cognitive and behavioral deterioration, consistent with classical Dravet phenotypes. These mutations failed to produce measurable sodium currents in a mammalian expression system, indicating complete loss of channel function. A fourth patient, who harbored the mutation p.Met1267Ile, though presenting with seizures early in life, showed lower seizure burden and higher cognitive function, matching borderland Dravet phenotypes. In correlation with this, functional analysis demonstrated the presence of sodium currents, but with partial loss of function. In contrast, six bioinformatics tools for predicting mutational pathogenicity suggested similar impact for all mutations. Likewise, homology modeling of the secondary and tertiary structures failed to reveal misfolding. In conclusion, functional studies using patch clamp are suggested as a prognostic tool, whereby detectable currents imply milder phenotypes and absence of currents indicate an unfavorable prognosis. Future development of automated patch clamp systems will facilitate the inclusion of such functional testing as part of personalized patient diagnostic schemes.Mutations in the  Children develop normally during the first months but subsequently exhibit unusually severe febrile convulsions before the age of 12 months. Later, various kinds of drug-resistant seizures appear, with or without myoclonus, tending towards the development of status epilepticus, especially during febrile illness. Following the onset of epilepsy, developmental delay becomes evident. Cognition continues to deteriorate and ultimately leads to moderate to severe intellectual disability. In parallel with the cognitive decline, communication skills become impaired and autistic features develop, as well as severe behavioral problems [de novo mutations in the SCN1A gene, which encodes for the alpha subunit of the type I voltage-gated sodium channel (NaV1.1), essential for neuronal activity [Dravet syndrome  is a developmental and epileptic encephalopathy of early childhood with an ominous course (www.ileaactivity .status epilepticus and sudden unexplained death in epilepsy (SUDEP), and that commonly prescribed antiepileptic sodium channel blockers aggravate Dravet seizures [SCN1A gene is recommended in infants presenting with two or more severe febrile seizures before the age of 12 months [SCN1A genetic analysis is not always trivial.Prompt and precise diagnosis of Dravet is critical, due to the high risk of seizures . Hence, 2 months . NeverthV1.1 loss-of-function. According to this, severe mutations that result in complete loss of NaV1.1 function lead to Dravet syndrome, whereas mutations that only partially reduce NaV1.1 activity cause milder phenotypic variants. Included among the latter is GEFS+ (genetic epilepsy with febrile seizure plus), a childhood-onset epilepsy that is well controlled by antiepileptic drugs; and milder forms of Dravet syndrome [www.ilae.org), which manifest lower seizure burden, higher cognitive abilities and lower risk of status epilepticus  [It has been suggested that the severity of clinical phenotype correlates with the degree of Nasyndrome  (www.ila ICEGTC) , 8.de novo SCN1A nonsense mutations that result in haploinsufficiency allows for immediate Dravet prognosis. However, predicting the damaging effect and clinical significance of de novo SCN1A missense mutations is less clear. While roughly half of Dravet patients harbor nonsense mutations, as many as 43% of patients carry de novo missense SCN1A mutations, leaving prognoses uncertain and families and physicians puzzled [de novo SCN1A mutations, which are the focus of this report, the clinical outcome of inherited SCN1A mutations varies even among kin, probably due to the influence of modifier genes [Identification of  puzzled . Intereser genes –12.de novo SCN1A mutation in patients with early seizure onset, we examined the correlations between clinical presentation, functional ramifications of sodium currents and bioinformatics prediction of expected pathogenicity in four de novo SCN1A missense mutations. Our data indicate that current bioinformatics tools cannot predict the severity of epilepsy or cognitive outcome. In contrast, functional studies in a mammalian expression system demonstrate a correlation between the degree of NaV1.1 loss-of-function, seizure burden and cognitive outcome. Thus, we propose the use of automated patch clamp systems, which provide rapid and technically easier functional analysis, as part of a personalized prognosis and treatment scheme for patients with de novo SCN1A missense mutations.To address the diagnostic challenge of SCN1A gene, were selected for in vitro and in silico modeling and functional studies. Clinical diagnosis of Dravet syndrome was made based on: a) severe febrile seizures early in life, b) drug-resistant seizures (with or without myoclonus), and c) cognitive deterioration starting after the second year of life [Four patients with Dravet syndrome, treated at our pediatric neurology clinic and found to harbor missense mutations in the  of life .status epilepticus, antiepileptic drugs used, type of schooling and control of seizures at three time points: two and five years of age, and most-recent visit. Seizure control was defined as the number of seizures per month . Existing EEG records at ages two and five years (±6 months) were used for spectral analysis of the background activity, as described by Holmes et al, 2012 [The following clinical data were extracted from patient charts: age, gender, molecular diagnosis, age at onset of seizures, type of seizures, age at onset of febrile seizures, presence of febrile/afebrile al, 2012 . Two 30 In contrast to standard developmental assessment, in which assorted IQ or DQ scores are evaluated using different methods, we uniformly administered VABS (Vineland Adaptive Behavior Score- Vineland II) performed during regular visits in our neurology clinic. Standard scores for the complete test and for different domains , as well as V scores for subdomains and maladaptive behavior index were computed and standardized to age according to the Vineland II manual .The study was approved by the institutional review committee at the Sheba Medical Center (IRB- 4870-18-SMC). Subject or parental informed consent was not required since the data was extracted from patient charts while anonymity of the subjects was protected.V1.1 Na+ channel α subunit , inserted into pCDM8 vector, was generously provided by Prof. William A. Catterall . The plasmid was propagated in TOP10/P3 cells  for > 30 h at 30°C, to minimize spontaneous rearrangement. The entire coding sequence was sequenced after each propagation. Point mutations were introduced using Phusion High-Fidelity DNA Polymerase  using the following primers: for NaV1.1G177AV1.1S259R 5’CTGTGTTCTGTCTGAGAGTATTTGCTCTAATTGGGCTG and 5’CAGCCCAATTAGAGCAAATACTCTCAGACAGAACACAG; for NaV1.1M1267I 5’ CATTTTCATTCTGGAAATACTTCTAAAATGGGTGGCATATGGC and 5’ GCCATATGCCACCCATTTTAGAAGTATTTCCAGAATGAAAATG; and for NaV1.1Q1923R 5’ CTGCTGTCATTATTCGGCGTGCTTACAGACGCCACC and 5’ GGTGGCGTCTGTAAGCACGCCGAATAATGACAGCAG5’CTTATAAAAATTATTGCAAGGGCATTCTGTTTAGAAGATTTTACTTTCC and 5’GGAAAGTAAAATCTTCTAAACAGAATGCCCTTGCAATAATTTTTATAAG; for Na.Human Na2. For electrophysiological recordings, cells were transiently transfected with cDNAs encoding NaV1.1WT or mutants (2 μg) and GFP (0.5 μg) in 35mm plates using polyethylenimine . Recordings were made 2–3 days after transfection. NaV1.1WT was transfected with each of the mutants, and each recording session started by recording currents from NaV1.1WT in order to verify normal expression.HEK-293 (ATCC CRL-1573) cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 10 units/mL penicillin and 10μg/mL streptomycin , and grown at 37 °C with 5% COV1.1WT or mutants and cultured for 3 days.For Western blot analysis, cells in 60 mm plates were transfected with 6 μg cDNA of Na2, 3 KCl, 1 CaCl2, adjusted to pH 7.35 with NaOH. Chemicals were purchased from Sigma-Aldrich  or Fisher Chemical . The voltage dependence of activation was measured from a holding potential of -120 mV. Cells were depolarized for 20 ms to potentials ranging from -70 to +40 mV in 10 mV increments, and peak inward currents were measured. Conductance (G)-voltage relationships were determined from peak current (I) versus voltage relationships as G = I/(V—VRev), where V was the test potential and VRev was the extrapolated reversal potential. The voltage dependence of inactivation was measured from a holding potential of -120 mV. Cells were depolarized for 500 ms to potentials ranging from -90 to +50 mV in 10 mV increments, followed by test pulses to -10 mV. Recovery from inactivation was examined by applying a 20 ms conditioning pulse to 0 mV from a holding potential of -120 mV, followed by a recovery interval of variable duration (Δ1 ms) and a test pulse to 0 mV. To examine NaV1.1 channel availability, repetitive depolarizations to 0 mV (2 ms long), at 50 Hz, from a holding potential of -70mV, were used.Our electrophysiological recordings were performed as described previously  with smaV1.1 mutants by applying the specific NaV1.1 opener, Hm1a (250 nM) [V1.1 modulator N,N'-bis(2-methylbenzamide), also known as NaV1.1-Compound 3a [V1.1-Compound 3a was shown to increase the firing of fast-spiking interneurons in brain slices and its calculated partition coefficient (clogP of 3.48) is expected to support membrane permeability [V1.1-Compound 3a (30 μM), but the drug was washed out prior to recording to prevent a reduction in peak amplitude.We attempted a rescue of the function of Na(250 nM) , or by ppound 3a . NaV1.1-eability . Cells wV1.1-Compound 3a were purchased from Alomone Labs .Hm1a and NaV1.1 antibody  or Alpha 1 Na+/K+ ATPase , followed by incubation with HRP-conjugated goat anti-rabbit antibody . The signal was visualized by chemiluminescent detection using the ECL Detection System.Cells were homogenized in 0.32M sucrose supplemented with protease inhibitors , 1mM EDTA and 1mM PMSF, pH 7.4. Crude membrane preparation was produced by centrifugation at 17,000 x g for 90 min. The precipitate (pellet) was solubilized in 150 mM NaCl, 2% Triton X-100 supplemented with protease inhibitors, 1mM EDTA and 1mM PMSF, pH 7.4. A 35 mg aliquot of total protein was separated on Tris-acetate gel (6%) and transferred onto PVDF membrane. After blocking with 5% w/v nonfat dry milk in TBST, the membrane was incubated with anti-NaThe data are expressed as mean ± standard error. Normality was tested using the Shapiro-Wilk’s W test. Equal variance was confirmed using the Levene’s test followed by two-tailed Student’s t-test or repeated measures ANOVA, as appropriate. SigmaPlot 12.5  was utilized to calculate statistical measures.http://sift.jcvi.org), MutationAssessor [http://mutationassessor.org), PolyPhen-2 (Polymorphism Phenotyping v2) [http://genetics.bwh.harvard.edu/pph2), Condel (CONsensus DELeteriousness score) [http://bbglab.irbbarcelona.org/fannsdb), and PROVEAN  [http://provean.jcvi.org). In addition, we assessed the pathogenicity of seven control mutations, previously described as causing either Dravet, GEFS+ or febrile seizures [The effects of the nonsynonymous amino acid change in the four mutations was predicted using 5 bioinformatics tools: SIFT   (http://Assessor  (http://ping v2)  (http://s score)    . Regarding the other assessment tools, a higher score means a greater probability that the prediction of pathogenicity is accurate.Mutations with a score over the threshold for each bioinformatics tool were considered pathogenic. For MutationAssessor , a highehttps://swissmodel.expasy.org). The following templates from the PDB repository (https://www.rcsb.org/pdb) were used for alignment: 4dck1.a (NaV1.5) for C terminal mutation and 5xsy.1.A (NaV1.2) for the other mutations. Information regarding NaV1.1 protein (UniProtKB—P35498) domains and secondary structures were extrapolated from the UniProt Knowledgebase (http://uniprot.org).Homology modeling was performed by submitting mutant and wild-type sequences to the SWISS-MODEL Workspace , 5.3–10.8 years of age (mean 8.1), harboring the following is study . The locis study .status epilepticus; one patient (p.Gly177Ala) developed a hypoxic ischemic event with cerebellar stroke and prolonged encephalopathy during status epilepticus. Seizure burden differed at 2 years of age, and in particular at 5 years of age  had electroencephalography (EEG) recordings at around two and five years of age, suitable for background analysis during the awake state. The mean background frequencies and power band measured in left frontal F3 electrodes are presented in All patients were evaluated with Vineland Adaptive Behavior Scale (VABS), which enables the comparison of patient performance at different ages . While tSCN1A mutations, we transiently transfected wild-type (WT) and mutant NaV1.1 channels into heterologous human HEK-293 cells. The resulting sodium currents were measured using whole-cell voltage clamp. Three of the four missense mutations, p.Gly177Ala, p.Ser259Arg and p.Glu1923Arg, failed to produce detectable sodium currents, suggesting a complete loss of function  reported complete loss-of-function in 11 out of 19 Dravet missense mutations listed [SCN1A missense mutations produce detectable current with an amplitude of at least 100 pA [In this study, three of the mutations tested  indicated complete loss-of-function effect, while the p.Met1267Ile mutation demonstrated complex biophysical changes, likely to cause partial loss of function. The degree of s listed –36. Whils listed , 32, 35,s listed . Furtherde novo missense SCN1A mutations, using automated patch clamp systems, can be implemented as part of the personalized diagnostic procedure and may soon become as routine and straightforward as genetic analysis.We thus propose the use of functional tests, using patch clamp analysis, as part of the diagnosis process. To date, patch clamp experiments are not readily available for clinicians. However, automated patch clamp set-ups are widely used in drug discovery companies, offering rapid and simple functional analysis of ion channel activity . TherefoV1.1 mutants G177A, S259R and Q1923R by pharmacological chaperoning -derived neurons may be needed. Moreover, genetic background greatly affects disease severity, especially in the case of inherited missense utations . Indeed,utations , p.Arg16utations  p.Met185utations ), with mutations –47. ForeAssessment of disease severity is challenging, as seizure burden and cognitive abilities of Dravet patients change with age. We compared the frequency of seizures and EEG background activity of patients at two and five years of age. Additionally, we administered the VABS test which, unlike other IQ or DQ tests, allows comparison of adaptive daily functions at different ages, irrespective of developmental level and cooperation. VABS has previously been used in two studies assessing cognition and behavior in children with Dravet , 49. Thide novo SCN1A missense mutations. Future technological development of automated patch-clamp set-ups, suitable for clinical use, may facilitate the inclusion of electrophysiological tests as part of a personalized diagnosis and treatment scheme for patients carrying de novo SCN1A mutations.Our data indicate that, while currently available bioinformatics tools are insufficient for predicting the severity of epilepsy and cognitive outcome, functional studies in mammalian expression systems may foretell the severity of S1 TableMean background frequencies and power band in left frontal F3 montage are depicted. No EEG`s are available for the patient with the p. Glu1923Arg mutation.(PDF)Click here for additional data file.S1 FigV1.1 channels in the absence (black) or presence (red) of 250 nM Hm1a. Currents were elicited by depolarizations to 0 mV from a holding potential of -120 mV. We calculated the effect of Hm1a as the difference in peak amplitude and inactivation (current at the end of the pulse / peak current) before and after Hm1a application. Hm1a increased the peak amplitude of NaV1.1WT by 16.8 ± 6.44%, and reduced the inactivation by 28.4 ± 3.57%. Comparable effects were measured for NaV1.1M1267I with 21.13 ± 8% increase of peak amplitude and 21.2 ± 7% reduction in inactivation.Currents from HEK-293 cells transiently expressing Na(PDF)Click here for additional data file.S2 FigV1.1 expression in total membranes. The rightmost lane represents untransfected HEK-293 cell (-). The lower panel is an Na+/K+ ATPase loading control. The bar graph is a quantification of the normalized expression of three independent experiments. For each lane, NaV1.1 protein expression was first corrected to the relative expression of the Na+/K+ ATPase loading control. Next, in order to combine different experiments, the data were further normalized to the corrected expression of NaV1.1WT in each experiment.Representative immunoblots of Na(PDF)Click here for additional data file.S3 FigV1.1 modulator, N,N'-bis(2-methylbenzamide), also known as NaV1.1-Compound 3a. The drug was not included in the external recording solution to prevent a reduction in peak amplitude. (A) Representative set of sodium current traces from HEK-293 cells expressing NaV1.1WT, NaV1.1G177A, NaV1.1S259R or NaV1.1Q1923R. (B) Mean current-voltage (I-V) relationships of sodium current densities. NaV1.1WT n = 12; NaV1.1G177A n = 10; NaV1.1S259R n = 7; NaV1.1Q1923R n = 7. (C) Average current densities at -10 mV, with or without (Cnt) incubation with NaV1.1-Compound 3a.The effect of pharmacological chaperoning. The cells were incubated for 48-72h with 30 μM of the Na(PDF)Click here for additional data file.S4 FigV1.1WT and NaV1.1M1267I, not normalized to cell capacitance. (B) Persistent currents (% of peak currents) measured at the end of 20 ms depolarization to 0 mV.(A) Mean current–voltage (I–V) relationships of peak currents for Na(PDF)Click here for additional data file.S5 FigV1.1 .Homology modeling of WT  and mutant Na(PDF)Click here for additional data file.


Resistance to chemotherapy drugs (e.g. taxol) has been a major obstacle in successful cancer treatment. In A549 human lung adenocarcinoma, acquired resistance to the first-line chemotherapy taxol has been a critical problem in clinics. Sphingolipid (SPL) controls various aspects of cell growth, survival, adhesion, and motility in cancer, and has been gradually regarded as a key factor in drug resistance. To better understand the taxol-resistant mechanism, a comprehensive sphingolipidomic approach was carried out to investigate the sphingolipid metabolism in taxol-resistant strain of A549 cell (A549T).A549 and A549T cells were extracted according to the procedure with optimal condition for SPLs. Sphingolipidomic analysis was carried out by using an UHPLC coupled with quadrupole time-of-flight (Q-TOF) MS system for qualitative profiling and an UHPLC coupled with triple quadrupole (QQQ) MS system for quantitative analysis. The differentially expressed sphingolipids between taxol-sensitive and -resistant cells were explored by using multivariate analysis.p < 0.001 and VIP > 1), including 35 sphingomyelins (SMs), 14 ceramides (Cers), 3 hexosylceramides (HexCers), 4 lactosylceramides (LacCers) and 1 sphingosine. A significant decrease of SM and Cer levels and overall increase of HexCer and LacCer represent the major SPL metabolic characteristic in A549T.Based on accurate mass and characteristic fragment ions, 114 SPLs, including 4 new species, were clearly identified. Under the multiple reaction monitoring (MRM) mode of QQQ MS, 75 SPLs were further quantified in both A549 and A549T. Multivariate analysis explored that the levels of 57 sphingolipids significantly altered in A549T comparing to those of A549 (This study investigated sphingolipid profiles in human lung adenocarcinoma cell lines, which is the most comprehensive sphingolipidomic analysis of A549 and A549T. To some extent, the mechanism of taxol-resistance could be attributed to the aberrant sphingolipid metabolism, “inhibition of the de novo synthesis pathway” and “activation of glycosphingolipid pathway” may play the dominant role for taxol-resistance in A549T. This study provides insights into the strategy for clinical diagnosis and treatment of taxol resistant lung cancer. Lung cancer has been the leading cause of cancer mortality, and adenocarcinoma is its most prevalent form . PaclitaIt has been recognized that the dysregulated metabolic profile of cancer is linked to the chemoresistance . Cancer The LIPID MAPS internal standard cocktail  was purchased from Avanti Polar Lipids . It was composed of uncommon SPLs which include: 17-carbon chain length sphingoid base analogs C17-sphingosine [So (d17:1)], C17-sphinganine [Sa (d17:0)], C17-sphingosine-1-phosphate [S1P (d17:1)], C17-sphinganine-1-phosphate [Sa1P (d17:0)], the C12-fatty acid analogs of the more complex SPLs C12-Ceramide [Cer (d18:1/12:0)], C12-ceramide-1-phosphate [C1P (d18:1/12:0)], C12-sphingomyelin [SM (d18:1/12:0)], C12-glucosylceramide [GlcCer (d18:1/12:0)], and C12-lactosylceramide [LacCer (d18:1/12:0)].3COOH, MS grade), formic acid , ammonium acetate  and potassium hydroxide  were purchased from Sigma-Aldrich . The HPLC grade chloroform (CHCl3), isopropanol (IPA), as well as methanol (MeOH) were purchased from Merck . Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640 medium, Fetal Bovine Serum (FBS), Penicillin-Streptomycin (PS) were obtained from Gibco, New Zealand. Sodium dodecyl sulfate (SDS) and 3--2,5-diphenyltetrazolium bromide (MTT) were acquired from Acros, USA. Ultrapure water (18.2 MΩ) was supplied with a Milli-Q system .Acetic acid  was added, the mixture was placed into a shaking incubator at 37 °C for 2 h. Acetic acid was used to neutralize the mixture before the typical four-step extraction was carried out for the preparation of SPLs. Further details for extracting SPLs and sample preparation were the same as previously described m/z 316 ions, with accurate mass acquired by Q-TOF, m/z 316.3202 at 6.532 min and m/z 316.2850 at 6.874 min correspond to [C19H41NO2 + H]+ and [C18H37NO3 + H]+ respectively, facilitating assignment of sphinganine (Sa) d19:0 and phytosphingosine (PTSo) t18:1. Further fragmentation in MS/MS confirmed the identification, a consecutive loss of 3 hydroxy groups can be observed in the latter case, which is the characteristic cleavage of PTSo  respectively corresponding to the sphingoid base chain of SM (d18:1/22:1) and SM (d18:2/22:0) were observed  and SM (d18:2/22:0) as example, there are 2 peaks corresponding to 2:1 and Sm/z 262.25, 264.27, 266.28). In A549 QC samples, 29 Cers, including 20 dehydroceramides, 8 dihydroceramides (DHCers) and 1 phytoceramide (PTCer), were identified by comparing the MS information and retention time with those of SPLs in our previous study [Ceramides are prone to fragment into product ions corresponding to the sphingoid base backbone , were unambiguously identified. All these SMs were characterized with a C18 sphingoid base chain, among which d18:1 type takes the largest proportion. In the N-acyl side chain, the number of carbon ranged between 14 and 26, with an unsaturation degree up to 5. Notably, all the DHSMs with 21 or less carbons in the N-acyl chain are fully saturated, while the others (with more than 21 carbons in the N-acyl chain) can be detected together with their corresponding de-hydrogen form. Three highly unsaturated SMs  including SM (d18:1/24:3), SM (d18:2/24:2) and SM (d18:2/24:3), have been detected in the QC sample of A549 & A549T cells.Hexose-linked glycoceramide including galactosylceramide  and glucosylceramide (GluCer) were represented as HexCer. All the 6 HexCers and 6 LacCers were found with d18:1 sphingoid base backbone. Only one HexCer with N-acyl chain in odd carbon number, HexCer (d18:1/23:0) was identified in A549. Notably, among all the HexCers and LacCers, only d18:1/24:1 species were identified as glycoceramides with unsaturated N-acyl fatty chain.Seventeen sphingoid bases as well as the analogs were also successfully identified. The carbon number ranging from 14 to 19 and the degree of unsaturation falls between 0 and 2. Two PTSo with 3 hydroxyl groups, PTSo t19:2 and PTSo t16:1, have been discovered for the first time.MRM mode of UHPLC-QQQ MS could provide accurate and sensitive approach under a wide range for quantitative analysis of SPLs. As the accuracy of triple-quadruple is about 0.1 Da, the quantification of SPLs cannot be accurately achieved merely with a QQQ analyzer, especially when suffering the isotopic interferences. Every unsaturated SPL could be recognized as an isotope of another one with the same characteristic backbone but less degree of unsaturation. For instance, if the LC separation is incomplete, the content of Cer (d18:1/24:0) will be artificially high due to the interference of Cer (d18:1/24:1) Fig. . In thisThe quantitative results indicated that SMs account for the majority of all the SPLs in A549 and A549T, among which SMs with C16/C18/C22/C24 N-acyl side chain took the largest proportion of the total content. SMs with d18:1 sphingoid backbone are the most dominant species, which take 27 out of all the 41 quantified SMs  = 0.874, Q2 (cum) = 0.845; Fig. 2X (cum) = 0.880, R2Y (cum) = 0.999 with an excellent prediction parameter Q2 (cum) = 0.998, and the score plot showed good visual separation between A549 and A549T groups as well , that markedly contributes to the classification.PCA was used for the overview of SPL dataset and the spotting of outliers, and thereby pick out trends of grouping or separation. It was performed to visualize general clustering among A549, A549T and QC groups [Rell Fig. . A totalUsing the sphingolipidomic approach, we obtained the detailed sphingolipid profiles for human lung adenocarcinoma cell A549 and its taxol resistant strain A549T, and then performed quantification. We found A549 and A549T share all the same species of SPLs, among which SM (dehydrosphingomyelin and DHSM), Cer , HexCer, LacCer, and sphingoid base were identified as the major SPLs. In contrast to normal A549, decreasing levels of Cer and SM concomitant with increasing of glycosphingolipids represent the main SPL metabolic profile of A549T. Totally 35 SMs, 14 Cers, 3 HexCers, 4 LacCers, and 1 sphingosine are recognized as metabolic pathway related biomarkers.de novo synthesis pathway”.  and LacCers account for a large proportion of biomarkers in A549T. It has been observed that glucosylceramide synthase is up-regulated after drug intervention and suggests that glycolipids may be involved in chemotherapy resistance . For decde novo synthesis pathway” and “activation of glycosphingolipid pathway” played the dominant role for taxol-resistance, and the key enzymes related to the pathways may have been altered. These results provide evidence to unravel the mechanism of taxol resistance in A549T. The distinctive phenotype could facilitate clinical diagnosis of taxol-resistant adenocarcinoma and provide insights into targets for the development of new drug against taxol resistance.Evidences suggest that tumor microenvironment including the sphingolipidome plays an important role in cancer drug resistance. So far to our knowledge, there is no sphingolipidomic study on taxol-resistant A549 human adenocarcinoma cell line. Based on the comprehensive identification and accurate quantification of SPLs, decreasing of Cer, SM and sphingosine concomitant with increasing of HexCer and LacCer have been characterized as the metabolic profile of A549T. It indicated that “inhibition of the


Acinetobacter ursingii M3 and Asticcacaulis excentricus M6 are plant growth-inhibiting bacteria that reduce the yield of the duckweed Lemna minor. We report here the complete genome sequences of A. ursingii M3 and A. excentricus M6, sequenced using the PacBio RS II platform. Acinetobacter ursingii M3 and Asticcacaulis excentricus M6 are plant growth-inhibiting bacteria that reduce the yield of the duckweed Lemna minor. We report here the complete genome sequences of A. ursingii M3 and A. excentricus M6, sequenced using the PacBio RS II platform. Plant growth-inhibiting bacteria (PGIB), also called deleterious rhizobacteria, repress host growth without any disease symptoms other than reduced growth . Despite2–Acinetobacter ursingii M3 and Asticcacaulis excentricus M6, the PGIB strains of the duckweed Lemna minor. Previously, 22 distinct bacterial strains were isolated from L. minor RDSC 5512 using conventional culture methods, and these were separately cocultured with sterilized Lemna minor samples to examine the effect on host growth. Unlike the majority of the isolates, which had positive or neutral effects on the host, A. ursingii M3 and A. excentricus M6 decreased the weekly yield of duckweed by 10 to 20%  according to the manufacturer’s protocol. Sequencing was performed with a PacBio RS II platform  using a single-molecule real-time (SMRT) cell 8Pac version 3 and a DNA polymerase binding P6 kit (Pacific Biosciences). Approximately 12 µg of DNA was used for construction of a SMRT cell library. For the genomes of A. ursingii M3 and A. excentricus M6, we obtained 107,291 and 152,454 quality-filtered subreads , totaling 1,126 and 1,373 Mb, respectively. De novo assembly was performed using the Hierarchical Genome Assembly Process (HGAP) version 3.0 with default settings. Gene prediction and annotation were conducted with Rapid Annotations using Subsystems Technology .The genomic DNA of A. ursingii M3 and A. excentricus M6. A. ursingii M3 contained seven sets of 5S-23S-16S rRNA genes, while A. excentricus M6 has one and two sets of 5S-23S-16S rRNA genes in chromosome 1 and chromosome 2, respectively. Further investigation is needed to screen for candidate genes involved in their plant growth inhibition. To our best knowledge, this is the first report of a PGIB genome sequence.The genome assembly yielded three circular contigs each for A. ursingii M3 and A. excentricus M6 have been deposited at DDBJ/EMBL/GenBank under the accession numbers AP018824 to AP018826 (BioProject PRJDB7166) and AP018827 to AP018829 (BioProject PRJDB7167), respectively.The genome sequences of


Diffuse midline glioma (DMG), H3 K27M-mutant, is a new entity in the updated WHO classification grouping together diffuse intrinsic pontine gliomas and infiltrating glial neoplasms of the midline harboring the same canonical mutation at the Lysine 27 of the histones H3 tail.n = 140) and methylation (n = 80) profiling was performed depending on the material available, in order to assess the biological uniqueness of this new entity compared to other midline and hemispheric pHGG.Two hundred and fifteen patients younger than 18 years old with centrally-reviewed pediatric high-grade gliomas (pHGG) were included in this study. Comprehensive transcriptomic (Tumor classification based on gene expression (GE) data highlighted the similarity of K27M DMG independently of their location along the midline. T-distributed Stochastic Neighbor Embedding (tSNE) analysis of methylation profiling confirms the discrimination of DMG from other well defined supratentorial tumor subgroups. Patients with diffuse intrinsic pontine gliomas (DIPG) and thalamic DMG exhibited a similarly poor prognosis . Interestingly, H3.1-K27M and H3.3-K27M primary tumor samples could be distinguished based both on their GE and DNA methylation profiles, suggesting that they might arise from a different precursor or from a different epigenetic reorganization.These differences in DNA methylation profiles were conserved in glioma stem-like cell culture models of DIPG which mimicked their corresponding primary tumor. ChIP-seq profiling of H3K27me3 in these models indicate that H3.3-K27M mutated DIPG stem cells exhibit higher levels of H3K27 trimethylation which are correlated with fewer genes expressed by RNAseq. When considering the global distribution of the H3K27me3 mark, we observed that intergenic regions were more trimethylated in the H3.3-K27M mutated cells compared to the H3.1-K27M mutated ones.H3 K27M-mutant DMG represent a homogenous group of neoplasms compared to other pediatric gliomas that could be further separated based on the type of histone H3 variant mutated and their respective epigenetic landscapes. As these characteristics drive different phenotypes, these findings may have important implication for the design of future trials in these specific types of neoplasms.The online version of this article (10.1186/s40478-018-0614-1) contains supplementary material, which is available to authorized users. IDH1 mutation, and their astrocytic or oligodendroglial phenotypes which are often associated with additional specific genetic alterations such as ATRX mutations or 1p/19q co-deletion, respectively. The discovery of recurrent mutations in the histone H3 genes in pediatric high-grade glioma has definitively separated these gliomas from the ones seen in adults [H3F3A gene are exclusively found in the hemispheres, K27M/I mutations in several histone H3 variants genes are specific to midline tumors [Diffuse intrinsic pontine glioma and malignant midline gliomas have the worst prognosis of all types of malignant tumors in children and adolescents , 4, 10. n adults , 26. Whie tumors . The 201In pediatric brain tumors, location has however long been seen as a master driver of oncogenesis that could reflect their different cells of origin , 9. WhetH3F3AversusHIST1H3B, with respect to differentiation markers, oncogenic programs, response to therapy and evolution [Moreover, we have shown two distinct forms of diffuse intrinsic pontine gliomas according to the type of histone H3 gene mutated, volution . These mvolution  leading The purpose of this work was therefore to better characterize a large series of pediatric midline high grade gliomas from the (epi)genomic, transcriptomic and anatomic point of view in order to identify the respective influences of these parameters on their biology described by their gene expression, methylome, and clinical behaviour.Moreover, we compared the H3-K27me3 landscape between the two main subgroups of DIPG, H3.1-K27M and H3.3-K27M, in patient deriving cellular models.High-grade glioma cases were reviewed centrally to confirm the diagnosis according to the 2007 WHO classification and its 2016 update as previously described , 20.Specific immunostainings were performed to detect nuclear expression of the trimethylation mark at position K27 of the histone 3 tail  as well as nuclear expression of the K27M form of histone H3 .GSCs were derived from DIPG tumors at diagnosis as previously described . Brieflyn = 99). Histone H3 gene mutational status was determined by Sanger sequencing for H3F3A, HIST1H3B/C and HIST2H3C [Frozen tissue samples were obtained from 119 pediatric patients with brain tumors of WHO grade III and IV . The samples were collected at Necker Hospital . Complete follow-up information was available for 82.5% of patients  cultures derived from patient biopsies at diagnosis and matching primary tumors were analyzed similarly .n = 10), MYCN (n = 10) and PDGFRA/pedRTK1 (n = 10) subgroup tumors. For t-SNE analysis , 428,230 uniquely mapping autosomal probes in common between the 450 k and EPIC arrays were used. The input for the t-SNE calculation is 1-Pearson correlation, weighted by variance. Clustering analyses were performed using the beta values of the top 10,000 most variably methylated probes by standard deviation. Methylation probes in the heatmap representation were reordered by unsupervised hierarchical clustering using Pearson correlation distance and median linkage.DNA was extracted from tumors and genome-wide DNA methylation analysis was performed using either the Illumina HumanMethylation450 BeadChip (450 k) or EPIC arrays. DNA methylation analysis was performed with custom approaches as previously described , 23. DNAGene expression analysis was conducted on an Agilent platform as previously described  but usinRNA-seq was performed on 21 primary tumor samples. Libraries were prepared using the TruSeq stranded mRNA sample preparation kit according to the supplier recommendations and paired-end sequencing was conducted on Illumina NextSeq500 to generate a mean of 150 million reads of 75 base pairs by sample. Trimmed reads were then mapped using tophat2 (v2.1.0) and bowtie2 (v2.2.5) first to the reference transcriptome, then to the reference genome for the remaining reads. Genes with a row sum of raw counts over the studied samples equal to or below 10 were filtered out to remove non-expressed genes. We handled outliers as default using minReplicatesForReplace = 7 in DESeq function used to estimate size factors, dispersion and model coefficients. Distances between samples were computed by using ‘1-Pearson correlation coefficient’ as the distance measure. PCA and t-SNE analysis were performed on the 250 genes associated with the highest variance to keep the same proportion of genes selected with the microarray analysis. All samples were projected on the two first principal components computed with rlog transformation of the counts of the 120 genes with the highest standard deviation. Using Rtsne package (v 0.11), we applied t-SNE on the same data matrix with the Pearson correlation as a distance and the following parameters: theta = 0, perplexity = min(floor((ncol(rlog_VariableGenes)-1)/3), 30), check_duplicates = FALSE, pca = FALSE, max_iter = 10,000, verbose = TRUE, is_distance = TRUE.RNA-seq was also performed on 6 distinct GSC models using TruSeq stranded total RNA sample preparation kit according to the supplier recommendations (Illumina) and then processed similarly as primary tumors.ChIP-seq of H3K27me3 epigenetic modification was performed in 6 GSC models at Active Motif according to proprietary methods. The 75-nt sequence reads were generated on a Illumina NextSeq 500 platform, mapped using BWA algorithm and peak calling was performed using SICER1.1 algorithm [http://hgdownload.soe.ucsc.edu/admin/exe/macOSX.x86_64/). Heatmaps of H3K27me3 ChIPseq enrichment across genomic loci were calculated using deepTools version 1.5.11. ComputeMatrix was used with regions of either +/− 5 kb or +/− 10 kb around the center of the genomic intervals for ‘bound regions’ or TSS for differentially expressed genes, respectively. Heatmaps were plotted with or without k-means (k = 5) and their average profiles of ChIP-Seq enrichment in the same − 10/+ 10 kb genomic intervals were also generated for each k-means group. The bigWig files  were annotated with chipSeeker package using UCSC hg19 known gene annotation and visualizated by peakAnno and Vennpie.Merging of the 3 biological replicates of H3.1- or H3.3-K27M subgroups was performed using bigWigMerge tool  was calculated according to the Kaplan-Meier method and all survival function estimate comparisons were performed in PRISM software using a log-rank test. OS was calculated from the date of histo-radiological diagnosis until death of patient from disease or last contact for patients who were still alive.data not shown). Then, we selected a set of genes associated with the highest standard deviation for subsequent tumor classification analysis (n = 120). Gene Ontology over-representation analysis showed an enrichment of genes involved in brain development  and morphogenesis ; many homeobox genes belong to this later set of genes reflecting probably the differences in tumor location.We conducted microarray gene expression profiling of the single center cohort from Necker Enfants Malades hospital of 119 pHGG with histone H3 genotype previously determined either by Whole Genome Sequencing  or targeH3F3A, HIST1H3B/C and HIST2H3A/C were located within the K27M DIPG subgroup. These tumors all showed H3K27-trimethylation loss by immunohistochemistry  and led to a similar conclusion as the k-mean group 1 corresponded to H3-K27M and H3-wild type samples presenting H3K27-trimethylation loss whereas k-mean group 2 contained all other pHGG tumors  was performed to highlight the principal sources of variation among pHGG tumors  in comparison with the other contrasts  , as well as an upregulation of genes involved in ion transmembrane transport (1.6e-04) and apoptotic processes (5.9e-18) and an downregulation of genes linked to cell cycle (8.3e-11) and gliogenesis (6.87e-10) in the case of the comparison between H3-wild-type and H3-K27M tumors. Also, geneset enrichment analysis (GSEA) identified the ontology GO_oligodendrocyte_differentiation (Enrichment Score 0.70) enriched in upregulated genes in H3-K27M tumors and GO_Cerebral_cortex_neuron_differentiation (Enrichment Score − 0.79) enriched in genes upregulated in H3-G34R. Some of these biological processes were previously identified as significantly enriched in differentially expressed genes when comparing H3.1- and H3.3-K27M tumors [In agreement with these observations, a huge number of differentially expressed genes were identified between H3-K27M and H3-wild-type tumors as well as between H3-K27M and H3-G34R tumors (adjusted n = 119) using only location information and then both location and H3 mutation status for patient stratification  deriving from these same biopsies. The sample classification by unsupervised hierarchical clustering confirmed the previous result, with two main clusters corresponding to H3.3-K27M and H3.1/2-K27M samples Fig. . AdditioDNA methylation is a relatively stable component of the epigenome involved in the establishment and maintenance of distinct gene expression patterns. Consequently, we decided to evaluate if the different DNA methylation profiles were associated with distinct transcriptome profiles. PCA analysis of GE measurements by microarrays did not clearly discriminate H3.1 and H3.3 mutated tumor samples Fig. . Indeed,To complement the description of the epigenetic landscape of DIPGs, we decided to assess the direct epigenetic consequences of the K27M mutation in H3.1-K27M and H3.3-K27M tumors, i.e. the loss and redistribution of the trimethylation mark at position K27. We thus took advantage of 6 GSC models of different genotypes as an expandable source of material and interrogated the genome-wide distribution of this mark with ChIP-seq. We conducted a PCA analysis on all genomic regions showing enrichment for this mark and could again separate the samples based on the type of mutated histone Fig. . This imThe average signal of all H3K27me3 peak regions was equivalent in H3.1- and H3.3-K27M tumors . The results showed that upregulated genes in H3.1-K27M are associated with a lower average signal of H3K27me3 and vice et versa for downregulated genes  H3 K27M-mutant. But this implied that the histone H3-K27M would be a stronger driver of oncogenesis than location. We used a pHGG cohort at diagnosis to evaluate the similarity of these H3-K27M mutated tumors at both DNA methylation and gene expression levels and compared them to other pHGG tumor subgroups in order to support or question the new update of the WHO classification .vs. supratentorial location of the tumor in the brain. Alternatively, location may not be considered at the structural level  but rather at the embryological level (midline vs. hemispheres) thus unifying midline tumors. Accordingly, survival analyses highlighted a similarly poor prognosis for DIPG and thalamic tumors, either mutated or not for histone H3. The bad outcome of all midline gliomas with K27M mutations was also observed by Karreman et al. [. Taken together these data support the rationale to define the same treatment paradigms for both midline K27M tumors and DIPG.Tumor classification based on microarray gene expression profiling revealed that K27M mutated tumors, either thalamic or pontine, can be discriminated from all others. Consequently, the molecular subtype appears to influence more the gene expression profile than the infratentorial n et al. . Taken tPDGFRA and MYCN subtypes represent three homogenous groups distinct from K27M tumors.The stratification based on DNA methylation profiling of our pHGG population also supports the similarity between thalamic and pontine H3-K27M tumors. Our results are concordant with previous reports concerning the discrimination of G34R/V and K27M mutated tumors depending on DNA methylation , 23. Morversus 13.5 months and 30.5 versus 18 months, respectively. Survival analyses also pointed out a significantly better outcome of histone H3 wild-type non-thalamic midline tumors, which likely reflects that they may be less diffusely growing gliomas and could therefore be more amenable to surgical resection, or that they exhibit a behavior of low-grade gliomas.The DIPG median survival was similar to the large retrospective pHGG cohort recently analyzed by MacKay and collaborators . HoweverFinally, in the gene expression analysis some diffuse midline gliomas without any H3-K27M mutation are grouped with the H3K27M tumors. Interestingly, they all exhibit a loss of the H3K27me3 mark as well. Thus, defining the entity by the H3K27M mutation only may therefore be too restrictive. Further studies are needed to sort this issue, especially since diffuse pontine and thalamic malignant gliomas have a poor prognosis irrespective of the presence of an H3K27M mutation or not as also recently shown in the HERBY trial .H3F3A or HIST1H3B/C. The sole H3.2-K27M sample clustered together with H3.1-K27M tumors, as expected given that they are both canonical histone H3 with identical role in the cell [and coll. did not report this distinction between H3.1 and H3.3 mutated tumors using DNA methylation profiling. This difference might result from a 10 times smaller proportion of H3.1 mutated samples analyzed (8 out of 441 samples) hiding out the variability brought by these tumors in their huge dataset. In addition, we used a 7 times larger set of probes  that might have captured more variations in the overall pHGG DNA methylation landscape.Interestingly, our methylation profiling data showed a subclassification of DMG, H3 K27M-mutant into two subgroups according to the histone gene affected by the K27M substitution, i.e. the cell . Yet, ththe cell . Histonethe cell . MacKay It is assumed -and was recently demonstrated by Hoadley et al., that DNA methylation can reflect the epigenetic memory of cancer cell-of-origin . Indeed,As a whole, the distribution of the H3K27me3 marks along the genome is different, both at the quantitative and qualitative levels. Average level of trimethylation at K27 is similar in both subtypes since only a small number of loci are highly enriched in H3K27me3 in H3.1 K27M mutated tumors, whereas the majority of the regions presenting this epigenetic mark are associated with a higher signal in H3.3-K27M. These H3K27me3 variations among the two subgroups are associated with the modulations of gene expression, many more genes being repressed in H3.3-K27M tumors. Qualitatively, K-means clustering of the distribution of this mark identified 5 clusters of genic regions and 5 clusters of intergenic regions differentially trimethylated at position K27 in the two subgroups of DIPG. We show that among differentially expressed genes, levels of H3K27me3 are anti-correlated with gene expression in general. However, gene expression could not be strictly explained by the levels of H3K27me3 in all cases leaving the possibility of additional levels of regulation for gene expression in DIPG.Overall, we provide molecular and clinical evidence in favor of the unification of all midline K27M mutated tumors that was proposed in the 2016 WHO CNS classification based on their common driver mutation. As such, these gliomas need to be considered as a unique entity in future clinical trials. Further analyses on the biology of H3.3 and H3.1 mutated diffuse gliomas are required to explain the distinction we have reported so far; this could allow testing specific precision medicine approaches in these two subgroups of diffuse midline gliomas.Additional file 1:Table S1. Summary of the 215 pHGG samples analyzed by either gene expression microarray, RNA-seq or methylation array. (XLSX 9 kb)Additional file 2:Table S2. PCA weights in gene expression analysis of pHGG. Table containing the weights of the 2 first principal components of each of the 120 genes used for the analysis of gene expression of the pHGG by PCA  were used for k-means analysis (k = 2) and the results represented in the same PCA as in panel A. The samples were color-coded according to k-means results, symbols reflect the histone H3 mutational status and their location are indicated in the plot. C-D. t-SNE analysis of the GE profiles of 119 pediatric high-grade gliomas using the genes associated with the highest standard deviation (n = 120 genes). The tumors were color-coded according to their location (A) or mutational histone H3 status (B) as described in Fig. p-value< 0.01). (PDF 224 kb)Additional file 4:Table S3. Lists of differentially expressed genes in microarrays data from H3-K27M vs. H3-WT, H3-G34R vs. H3-K27M, H3-G34R vs. H3-WT comparisons . (XLSX 174 kb)Additional file 5:Figure S2. Principal component analysis of the GE profile of 21 DIPG using the gene associated with the highest standard deviation . (PDF 54 kb)Additional file 6:Figure S3. A-C. Metaplots showing average signal accumulation in reads of all the regions bound by H3K27me3 in at least one sample  or H3K27me3 occupied regions with or without overlapping genes  in both H3.1- and H3.3-K27M GSC cells. Each plot is centered on the summit of the average occupancy and extended 10 kb upstream and downstream . Below the metaplots, heatmaps illustrating average H3K27me3 levels in the 20 kb genomic intervals centered on the summit of the peak in each subgroup are presented. D. Violin plot displaying transcript expression level of RNA-seq data in tpm in H3.1- and H3.3-K27M subgroups. Boxplots represent the 5th,25th,75th and 95th percentiles and the median of the transcript distribution, The distributions were divided in 4 categories: non expressed genes (< 0.1 tpm), low expressed genes (from 0.1 to 1 tpm), intermediate (from 1 to 10 tpm) and highly expressed genes (> 10 tpm). (PDF 2819 kb)


Adenosine-to-inosine (A-to-I) RNA editing, mediated by ADAR1 and ADAR2, occurs at tens of thousands to millions of sites across mammalian transcriptomes. A-to-I editing can change the protein coding potential of a transcript and alter RNA splicing, miRNA biology, RNA secondary structure and formation of other RNA species. In vivo, the editing-dependent protein recoding of GRIA2 is the essential function of ADAR2, while ADAR1 editing prevents innate immune sensing of endogenous RNAs by MDA5 in both human and mouse. However, a significant proportion of A-to-I editing sites can be edited by both ADAR1 and ADAR2, particularly within the brain where both are highly expressed. The physiological function(s) of these shared sites, including those evolutionarily conserved, is largely unknown.Adar1E861A/E861AIfih1−/−) with rescued ADAR2-deficient (Adarb1−/−Gria2R/R) animals. Unexpectedly, the global absence of editing was well tolerated. Adar1E861A/E861AIfih1−/−Adarb1−/−Gria2R/R were recovered at Mendelian ratios and age normally. Detailed transcriptome analysis demonstrated that editing was absent in the brains of the compound mutants and that ADAR1 and ADAR2 have similar editing site preferences and patterns.To generate completely A-to-I editing-deficient mammals, we crossed the viable rescued ADAR1-editing-deficient animals (We conclude that ADAR1 and ADAR2 are non-redundant and do not compensate for each other’s essential functions in vivo. Physiologically essential A-to-I editing comprises a small subset of the editome, and the majority of editing is dispensable for mammalian homeostasis. Moreover, in vivo biologically essential protein recoding mediated by A-to-I editing is an exception in mammals. Adar) and ADAR2 (Adarb1). The third mammalian ADAR, ADAR3 (Adarb2), does not have detectable editing activity .Sequenced reads (150 bp) were trimmed for adaptor sequence and low-quality reads using fastp (v 0.19.5) . ParametFor transcriptome analysis, trimmed reads were aligned using Salmon  (versionhttp://victorian-bioinformatics-consortium.github.io/degust/). Briefly, genes were only considered with count > 3 and CPM > 1 in at least 3/3 samples of a given genotype. Normalized read counts (moderated log counts per million) and differential expression were generated using edgeR .Trimmed reads were aligned to the MM10/GRCm38 reference genome with transcript annotation (gencode.mm10.vM14.annotation.SEQINS.gtf) with STAR (version 2.6.0c)  using thA database of 57,077 murine editing sites was compiled from published databases (RADAR ), publichttps://github.com/dieterich-lab/JACUSA): parameters used: -F 1024 -filterNH_ 99, -filterNM_ 99, -c 3 -P RF-FIRSTSTRAND. Briefly, call-1 was used to determine the RNA editing level for all known sites for each individual sample replicate. Duplicate reads were removed. For sites not called by JACUSA, we added read depth calculated by samtools pileup to reflect the sequence coverage at those positions. The editing rate for each genotype was calculated as the sum of edited reads for three replicates/total read depth for all three replicates. Sites required ≥ 50 read coverage in all samples  of the comparison and an editing rate of ≥ 0.01 (≥ 1%) in the WT and dHet to be considered. See Additional file Editing calling of known sites (RNA vs mm10) was performed using JACUSA 2.0.0-RC5 (70)  using Goldmine . B1 and Sequence logos were generated using ggseqlogo .GSE132214 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE132214).All datasets related to this work are deposited in GEO. Dataset accession number: P < 0.05 was considered significant. Data are presented as mean ± SEM unless otherwise stated.For biological experiments, the significance of results was analyzed using the one-way or two-way ANOVA with multiple comparison corrections unless otherwise stated; calculated as Prism software, Additional file 1: Figure S1 . Comparison of gene expression signatures by genotype; data from Panel 3A. Analysis of transcriptional signatures in the 12 week old male brain of each genotype. n=3 independent samples per genotype. The increased expression of the transcripts highlighted in blue is shared between murine and human ADAR1 mutants. (A) Y-axis has the gene expression comparison of the Adar2-/- vs the dHet; x-axis has the gene expression comparison of the Adar1E861A/E861A vs WT. Gene expression changes dependent on Adar1 loss occur on the x axis, those dependent on the loss of Adar2 on the y axis. (B) Adar2-/-compared to Adar1E861A/E861A Adar2-/- (dKO); (C) Adar1E861A/E861A compared to Adar1E861A/E861A Adar2-/- (dKO). Figure S2. Comparison of the gene expression signatures by genotypes; data derived from comparisons in Panel 2A. Figure S3. Altered sites identified in analysis of Adar1E861A/E861A Adarb1-/- (dKO); related to Panel 4B. Analysis of sites identified as altered compared to ref seq or batch control in the dKO samples. Individual sites with IGV screenshots and the full list of sites with variants identified in analysis of the double KO samples.Additional file 2 Dataset S1. Full histopathology report from Adar1E861A/+Ifih1-/-Adarb1+/-Gria2R/R (dHet) and Adar1E861A/E861AIfih1-/-Adarb1-/-Gria2R/R (dKO).Additional file 3: Dataset S2. RNA-seq data used for differential gene expression analysis. Samples=12 week old male whole brain; n=3 per genotype. Related to Fig Additional file 4: Dataset S3. QuSAGE pathway analysis of gene expression datasets. Samples= 12 week old male whole brain; n=3 per genotype. Related to Fig Additional file 5: Dataset S4A. Editing analysis of the known sites. Related to Fig. Additional file 6:Dataset S4B. De novo discovery of RNA editing sites in each genotype using JACUSA2.0.0 (transcriptome comparison to C57Bl/6 reference genome). Related to Fig Additional file 7:Dataset S5. ADAR1 and ADAR2 specific editing events – frequency of editing. Related to Fig. Additional file 8: Review history.