These replication-inducible VACVs would only express the selected genes in the presence of tetracyclines. VACVs inducibly expressing the A3L or A6L genes replicated indistinguishably from wild-type VACV in the presence of tetracyclines, whereas there was no evidence of replication in the absence of antibiotics. These outcomes were reflected in mice, where the VACV inducibly expressing the A6L gene caused weight loss and mortality equivalent to wild-type VACV in the presence of tetracyclines. In the absence of tetracyclines, mice were protected from weight loss and mortality, and viral replication was not detected. These findings indicate that replication-inducible VACVs based on the conditional expression of the A3L or A6L genes can be used for the development of safer, next-generation live VACV vectors and vaccines. The design allows for administration of replication-inducible VACV in the absence of tetracyclines (as a replication-defective vector) or in the presence of tetracyclines (as a replication-competent vector) with enhanced safety.Vaccinia virus (VACV) has been used extensively as the vaccine against smallpox and as a viral vector for the development of recombinant vaccines and cancer therapies. Replication-competent, non-attenuated VACVs induce strong, long-lived humoral and cell-mediated immune responses and can be effective oncolytic vectors. However, complications from uncontrolled VACV replication in vaccinees and their close contacts can be severe, particularly in individuals with predisposing conditions. In an effort to develop replication-competent VACV vectors with improved safety, we placed VACV late genes encoding core or virion morphogenesis proteins under the control of  Vaccinia virus (VACV) is the prototype member of the genus Orthopoxvirus within the family Poxviridae, which also includes Cowpox virus, Monkeypox virus, and most notably Variola virus, the causative agent of smallpox. Immunization with VACV led to the successful eradication of smallpox worldwide [orldwide  and sincorldwide –4. Howevorldwide –9 can reorldwide .A number of strategies have enhanced the safety of VACV vectors. These include the selection of natural strains with lower virulence, the development of highly-attenuated strains such as modified vaccinia Ankara (MVA) and NYVAC , 13, ina10 operon that confers tetracycline resistance in bacteria. Tetracyclines are broad-spectrum antibiotics that inhibit translation in gram-positive, gram-negative, and atypical bacteria [tet operon, the Tet repressor (TetR) is unable to bind to tet operators in the presence of tetracyclines, allowing transcription of the tetracycline resistance gene [tet operon has been adapted to a variety of organisms for inducible gene expression, including prokaryotic, yeast, insect, plant, and mammalian cells, as well as transgenic organisms and viruses [tetR) constitutively and inserting a tet operator element (O2) after the transcriptional start site of VACV genes, allowing their expression to be regulated by tetracyclines [in vivo by using tetR and tetO2 to control the transcription of genes that are required for viral replication. Ideally, these replication-inducible VACVs should replicate to wild-type levels in the presence of tetracyclines such as doxycycline (DOX) and would be unable to replicate in the absence of these antibiotics.In a continuing effort to improve VACV vectors, a new approach is to develop replication-inducible VACV vectors that are significantly safer yet replicate to the same levels as their parental strains, and therefore maintain their full immunogenic and oncolytic potential. These replication-inducible VACV vectors are based on elements of the transposon Tnbacteria . In the nce gene . The tet viruses –32. Contcyclines , 34. TheOur previous work on tetracycline-dependent expression of VACV early transcription factors  led us ttet operon to design and construct VACVs that inducibly express the E8R, A3L, or A6L genes. We evaluated and characterized the properties of these VACVs and showed that viruses inducibly expressing A3L, A6L, or E8R replicate indistinguishably from wild-type VACV in the presence of tetracyclines, and more importantly, that viruses inducibly expressing A3L or A6L only abortively infect cells in the absence of antibiotics. Furthermore, a VACV inducibly expressing A6L was not detected in mice infected in the absence of tetracyclines but caused weight loss and mortality similar to wild-type VACV in the presence of tetracyclines.We used elements of the tetR gene under a constitutive VACV promoter and incorporating tetO2 immediately downstream from the promoters directing the expression of the E8R, A3L, or A6L genes  of these genes as shown in 11) late promoter, which has been used successfully as a lac-responsive promoter [11A6L for VACV inducible A6L using the P11 promoter) were expected to replicate only in the presence of tetracyclines such as DOX.Replication-inducible recombinant VACVs were designed by expressing the 6L genes . VACV lapromoter , was useEscherichia coli xanthine-guanine phosphoribosyl transferase (gpt) gene and the screening marker enhanced green fluorescent protein (EGFP) gene as a fusion gene (gpt-EGFP) under the control of the synthetic strong early/late VACV promoter PE/L [tetR under another (back-to-back) PE/L promoter, a tet-responsive promoter  to control the expression of the target genes, and left and right recombination sequences  virus. In addition, PCR analysis of viral DNA purified from high-titer stocks with multiple primers spanning the regions of interest , followed by crystal violet staining 2 or 7 days post-infection (DPI). Isolated plaques that formed 2 DPI in the presence of DOX by viE8R, viA3L, and viP typical  and iden typical , suggeste of DOX . Moreoveen 7 DPI , when th11A6L, only single EGFP+ cells could be detected in the absence of DOX  of 0.01 in the absence or presence of multiple concentrations of DOX by determining virus yield 48 hpi. As expected, the wild-type WR strain replicated equally in the absence or presence of DOX . Additio11O2 promoter used to develop viP11A6L (E8RO2 and PA3LO2 promoters in the transfer vectors (11O2 promoter sequence (11E8R) or A3L (viP11A3L) under P11O2.The putative natural promoter sequences present in the upstream intergenic regions were used to control the expression of the E8R and A3L genes in viE8R and viA3L, respectively . Since vviP11A6L . This wa vectors  with thesequence . New rec11E8R and viP11A3L produced plaques similar to WR in the presence of DOX , or no plasmid (mock). Complementation was assessed by measuring virus yield in the absence or presence of the complementing plasmids. Cells infected with viP11A6L and transfected with pP11A6L yielded approximately a two-log increase in virus titers when compared to cells transfected with pP11A3L or no plasmid, although yield was not as high as virus grown in the presence of DOX expressing the A6L gene directly from the genome  [11A6L survived infection without weight loss or other clinical signs, with significantly higher mean weights than all other groups from 2–8 DPI. In the presence of DOX, viP11A6L-infected mice exhibited weight loss, clinical symptoms , and mortality indistinguishable from WR   in the a from WR .11A6L to the parental WR in tissue, groups of five CB6F1/J female mice were infected intraperitoneally with ~2 × 106 PFU viP11A6L or WR, in the absence or presence of 0.125 mg/ml DOX in drinking water. Viral loads in ovaries were determined by plaque assay 6 DPI .The replication-inducible VACVs were designed to control the expression of genes encoding core or virion morphogenesis proteins. The O11A6L does not form plaques in the absence of DOX, even when cells were incubated for 7 days. Singly-infected cells could be detected by expression of the EGFP reporter protein 2 DPI, and the neighboring cells did not show any evidence of infection such as CPE or EGFP expression. High levels of EGFP expression in these abortively-infected cells suggest that gene expression from the PE/L promoter was not compromised in the absence of DOX. In the presence of DOX, viP11A6L formed typical plaques, even when DOX was added 2, 4, or 6 DPI, indicating that the virus growth cycle resumed once A6L gene expression was allowed. Replication did not resume when DOX was added 8 or 10 DPI, possibly because the abortively-infected cells or the virus life cycle were compromised this late after infection. Additionally, viP11A6L did not form plaques in the absence or presence of 1 ng/ml DOX, and plaque sizes were indistinguishable from wild-type VACV at ≥10 ng/ml DOX. viP11A6L replicated minimally at 1 ng/ml and virus yield at ≥10 ng/ml DOX was indistinguishable from WR, demonstrating that viP11A6L replicates to wild-type levels under these conditions.We showed that viP11 (F17R) promoter sequence, used successfully in viP11A6L, to generate viP11E8R and viP11A3L. However, in the absence of tetracyclines viP11E8R still formed plaques  and showed evidence of replication. Conversely, no plaques were detected in viP11A3L-infected cell monolayers in the absence or presence of 1 ng/ml DOX, and plaques comparable in size to WR were formed in the presence of 100 ng/ml DOX. Notably, there was no evidence of viP11A3L replication in the absence of DOX, and virus yield at 100 ng/ml DOX was indistinguishable from WR. Additionally, transient complementation confirmed that inducible replication of viP11A3L and viP11A6L was achieved through control of A3 and A6 expression. viP11A6L was further tested in vivo, where weight loss and mortality were observed after intranasal infection in the presence of only 0.125 mg/ml DOX in drinking water. Importantly, mice inoculated with viP11A6L in the presence of DOX exhibited similar levels of weight loss, mortality, and viral replication as mice inoculated with wild-type (WR) VACV, but did not result in any clinical signs in the absence of DOX.Since viE8R and viA3L replicated even in the absence of tetracyclines, it is possible that control of the native E8R and A3L promoters was unsuccessful because their sequences were not identified correctly. Thus, the putative natural E8R and A3L promoter sequences were replaced with the Ptet operon elements, the virus replicated both in the absence and presence of tetracyclines. Interestingly, viP11E8R plaques formed in the absence of inducer were significantly smaller than wild-type VACV plaques. This suggests leaky expression of E8R in the absence of tetracyclines, or that the E8R gene is not essential for viral replication in vitro.The E8R gene has been studied extensively, although its specific function remains unclear –39. In olac operon elements in a system where the bacteriophage T7 RNA polymerase is expressed under a late promoter controlled by a lac operator, and the A3L gene is placed under a T7 promoter also controlled by a lac operator [The function of A3 has been studied previously using temperature-sensitive mutants where it was found that virions formed at non-permissive temperatures were abnormal in shape, had substantially reduced infectivity, and reached transcription levels less than 2% of the wild-type virus, thus indicating A3 plays a critical role in virion morphogenesis . Additiooperator . In the lac operon elements [lac operon-based virus expressing A6L was shown to replicate only in the presence of IPTG, although about 10-fold less efficiently than the parental virus [11A6L was able to replicate indistinguishably from wild-type VACV in the presence of DOX both in vitro and in vivo, suggesting that our genetic modifications did not attenuate the virus.The function of A6 has been previously studied in VACVs with epitope-tagged A6 and temperature-sensitive mutants , as wellelements , 65. Theelements , 49, 65.gpt and EGFP) could be developed by transient dominant selection or other methods [In future studies, the safety of replication-inducible vectors can be confirmed in immunocompromised or other more rigorous immunodeficient  mouse models by evaluating weight loss, clinical signs, mortality, and replication in the presence of DOX. In addition, the immunogenicity of the inducible vectors can be compared to wild-type and parental viruses to make sure that they remain fully immunogenic, and therefore maintain their vaccine and therapeutic efficacy, even under antibiotic treatment. Further modifications to the vectors  would enhance their use as oncolytic viruses. Finally, marker-free versions of these replication-inducible vectors  was obtained from ATCC (VR-2035) and a clone (9.2.4.8) derived by sequential plaque purification was used to generate the recombinant viruses in this study [Cell lines were obtained from the American Type Culture Collection . African green monkey BS-C-1 (CCL-26) and human HeLa S3 (CCL-2.2) cells were grown in Dulbecco’s modified Eagle medium  supplemented with 10% tetracycline-tested fetal bovine serum , MEM vitamin solution, 200 mM L-glutamine, and MEM non-essential amino acids . All cells were grown at 37°C in 5% COis study . High-ti1/J) mice (stock #100007) were purchased from Jackson Laboratory and maintained in accordance with animal care protocols approved by the Institutional Animal Care and Use Committee at the University of Connecticut (Protocol Number A16-029). All inoculations were performed under isoflurane anesthesia. Weight, clinical signs , and mortality were assessed daily. Euthanasia (weight loss ≥ 25%) was performed by carbon dioxide overdose followed by cervical dislocation.Five-week-old female (BALB/cJ × C57BL/6J) normal hybrid  and the EGFP gene , using a previously developed strategy [tetR gene (based on GenBank:X00694) was synthesized with an internal VACV early transcriptional termination sequence (TTTTTNT) removed from the middle of the gene (Leu codon at position 358 changed from TTA to CTT) to ensure early gene expression. The tetR and gpt-EGFP genes were placed under back-to-back PE/L synthetic promoters (sequence TATTTATATTCCAAAAAAAAAAAATAAAATTTCAATTTTTAACTGCAGTTAAAAATTGAAATTTTATTTTTTTTTTTTGGAATATAAATA) [11 late VACV promoter with a tet operator (O2) [The schematic representation of the VACV transfer vector backbone used for the generation of the recombinant VACVs is shown in strategy . The tetATAAATA) . The trator (O2)  placed igpt-expressing VACVs were plaque purified from transfection lysates in BS-C-1 cells using selection medium  [+ plaques were visualized under a Carl Zeiss Axio Observer D1 inverted fluorescence microscope  using an XF100-2 (EGFP) filter . All VACVs were plaque purified at least four times to eliminate contamination with the parental virus. High-titer stocks were generated by infecting HeLa S3 cells with the VACVs at an MOI of 0.1 in the presence of 1 μg/ml DOX. Infected cells were harvested 4 DPI by centrifugation at 300 × g for 10 min and resuspension in D-MEM without tetracyclines. Cells were then lysed by freezing and thawing, sonicated, and trypsinized. Finally, cell lysates were clarified to remove contaminating cell debris by a second round of sonication and centrifugation at 500 × g for 10 min. For use in inoculation, these stocks were amplified in HeLaS3 cells, processed as described above, then pelleted through a 36% sucrose cushion [Recombinant VACVs were generated by standard homologous recombination after transfection of the transfer vectors with FuGENE HD transfection reagent  into BS-C-1 cell monolayers infected 2 h previously with VACV WR clone 9.2.4.8 at an MOI of 0.05. Recombinant anthine)  in the p cushion .To detect any residual parental VACV after plaque purification, EGFP expression was confirmed by fluorescence microscopy. Briefly, plaque assays were performed on BS-C-1 cell monolayers in 6-well plates in the absence or presence of 1 μg/ml DOX using high-titer stocks. After 2 days of incubation at 37°C, plaques were analyzed by both brightfield and fluorescence microscopy to detect any EGFP-negative plaques that could be present and would represent unstable recombinants or recombinants needing further plaque purification. The genomic organization of each recombinant VACV around the insertion site was checked by PCR analysis of viral DNA purified using a small-scale method employing micrococcal nuclease . The priThe ability of the VACVs to replicate in the absence or presence of inducer (DOX) was investigated by standard plaque assay. Briefly, near-confluent BS-C-1 cell monolayers in 24-well plates were infected with the VACVs at approximately 5–20 plaque-forming units (PFU)/well in the absence or presence of 1 μg/ml DOX and incubated at 37°C for 2 or 7 days. Cells were stained and fixed with 0.5% crystal violet in 10% ethanol/20% formaldehyde and isolated viral plaques were imaged with a digital camera or an inverted microscope.For the analysis of plaque formation by fluorescence microscopy, near-confluent BS-C-1 cell monolayers in 24-well plates were infected with the VACVs at 5–20 PFU/well in the absence or presence of 1 μg/ml DOX. Plaques and infected cells were imaged at 2, 4, 6, 8, 10, and 12 DPI. In a subset of wells infected with the VACVs in the absence of inducer, DOX was added at 2, 4, 6, 8, or 10 DPI and any plaques that formed were imaged 2 days later.The size of the plaques formed by the VACVs in the absence or presence of DOX was investigated by plaque assay. Briefly, near-confluent BS-C-1 cell monolayers in 12-well plates were infected with the VACVs at 30 PFU/well in the absence or presence of 1, 10, 100, or 1000 ng/ml DOX and incubated at 37°C for 36 h. Cells were stained and fixed with 0.5% crystal violet in 10% ethanol/20% formaldehyde and the radius of isolated plaques was measured under an inverted microscope with measurement-capable software .g to clarify. Virus titers were then determined by a plaque assay on BS-C-1 cells (in duplicate) in the presence of 1 μg/ml DOX.Triplicate monolayers of near-confluent BS-C-1 cells were infected with the VACVs at an MOI of 0.01 in 24-well plates in the absence of tetracyclines. After 1 h, supernatants were aspirated and replaced with medium containing 0, 1, 10, 100, or 1000 ng/ml of DOX. Immediately after this step (to determine input titer) or after 48 h of incubation at 37°C (to determine virus yield), cells were scraped, centrifuged, and resuspended in 0.5 ml of D-MEM. Samples were processed in three cycles of freeze/thaws and sonication, followed by trypsinization, sonication, and centrifugation for 10 min at 500 × 11 late promoter  were generated by PCR cloning with primers 11–12 (A6L) or 13–14 (A3L) , or no plasmid (mock). Cells were incubated for 48 h at 37°C in the absence or presence of 1 μg/ml DOX, then scraped, centrifuged, and resuspended in 0.5 ml of D-MEM. Virus titers were then determined by a plaque assay on BS-C-1 cells (in duplicate) in the presence of 1 μg/ml of DOX.Plasmids expressing the A6L or A3L genes under the VACV P14 (A3L) . Primers1/J mice were established based on average body weight over three consecutive days. Each group was formed so that mean body weight and standard deviation between groups were approximately equal. At 1 day prior to infection, DOX was filter-sterilized and diluted in autoclaved water at 2, 0.25, 0.125, 0.025, and 0.005 mg/ml, and provided ad libitum to the appropriate group [4 PFU viP11A6L in a final volume of 20 μl sterile PBS. Weight, clinical signs and mortality were assessed daily until 14 DPI. Animals were euthanized by CO2 overdose and cervical dislocation if weight loss ≥ 25%.Groups of five CB6Fte group . DOX wat1/J mice were established, and 0.125 mg/ml DOX in drinking water was provided to appropriate groups as described above. On day 0, groups were inoculated intranasally (~10 μl per naris) with a lethal dose of ~2 × 106 PFU virus (viP11A6L or WR) in a final volume of 20 μl sterile PBS. Weight, clinical signs and mortality were assessed daily until 14 DPI. Animals were euthanized by CO2 overdose and cervical dislocation if weight loss ≥ 25%.To evaluate weight loss and survival, groups of 10 CB6F1/J mice were inoculated intraperitoneally with ~2 × 106 PFU viP11A6L or WR, in the presence or absence of DOX. On day 6 post-infection, mice were euthanized and ovaries collected for processing. Ovaries were weighed and homogenized in 10% volume D-MEM by weight. Homogenates were processed in three cycles of freeze/thaws and sonication, followed by trypsinization, sonication, and centrifugation for 10 min at 500 × g to clarify. Supernatants were used to titrate virus in 6-well plates of near-confluent BS-C-1 cells in the presence of 1 μg/ml DOX.To assess viral replication in ovaries, groups of five CB6FStatistical analyses were performed with GraphPad Prism v. 7.0c . Images were processed in Adobe Photoshop CS6  with no manipulations other than for contrast.


Drosophila melanogaster dopamine transporter (dDAT) (PDB 4M48 and PDB 4XPA) as templates, we developed molecular models for norepinephrine (NE) bound to its high affinity binding site (S1) in the hNET. Our model suggests that the S1 site for NE is deeply buried between transmembrane helices (TMHs) 1, 3, 6, and 8 and overlaps the binding site for leucine in the bacterial leucine transporter (LeuT) and dopamine (DA) in dDAT. Mutational studies identified the functional binding pocket for NE comprised residues A73, A77, N78, V148, N153, I156, G320, F329, N350, S420, G423, and M424, which all influenced NE affinity and/or transport. These effects support a NE-hNET docking model where A73, A77, G320, S420, G423, and M424 form H-bond interactions with NE, V148, I156, and F329 form hydrophobic interactions with NE, whereas N78 affects NE transport and N350 affects NE affinity and transport via an influence on the octahedral co-ordination of the Na1+ ion. Consistent with a conserved structure-function amongst sodium-dependent neurotransmitter transporters, S1 residues A73, A77 (G100 in hSERT), N78, V148 (I150 in hSERT), N153, G320, F329 (Y331 in d DAT), N350, and G423 are conserved in DAT and SERT, indicating they likely play conserved functional roles.The human norepinephrine transporter (hNET) is a member of the neurotransmitter/sodium symporter family, which also includes the neuronal monoamine transporters for serotonin (SERT) and dopamine (DAT). Its involvement in chronic pain and many neurological disorders underlies its pharmaceutical importance. Using the X-ray crystal structures of the human serotonin transporter (hSERT) (PDB 5I6X) and  Spatio-temporal regulation of NE neurotransmission, presynaptic homeostasis and maintenance of extra synaptic monoamine levels is maintained by the NET . The NETAquifex aeolicus) LeuT  also helped identify a second low affinity binding site (S2) of NE at hNET (Drosophila melanogaster dopamine transporter (dDAT) bound to the tricyclic antidepressant nortriptyline was solved at 2.95 Å  LeuT . This st at hNET . More ret 2.95 Å  and dDATt 2.95 Å . Interest 2.95 Å . The amit 2.95 Å . In 2016t 2.95 Å  revealedIdentification of functionally significant ligand interactions have mostly focused on the inhibitor site, with residues affecting cocaine  and its 3H]-NE (40–80 Ci/mmol), [3H]-NX (70–87 Ci/mmol), OptiPhase Supermix cocktail and Betaplate Scintillant were purchased from PerkinElmer Life Sciences .Dulbecco’s Modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from Invitrogen . Cell dissociation buffer was purchased from Life Technologies. Nisoxetine (NX), mutation and sequencing primers were purchased from Sigma-Aldrich. Cell culture dishes and 96-well plates were purchased from Nunc .  for 1 h. The primary sequence-specific antibody  was added in 1:3000 dilution in blocking solution and incubated for 45 min on a shaker. Excess antibody was then removed by washing three times in blocking solution for 5 min each. The secondary antibody  was then added in 1:2500 enzyme to blocking solution and allowed to bind for 45 min. The excess secondary antibody was removed with three 1 min washes with PBS. The membrane was scanned using Odyssey IR imaging system (LI-COR Biosciences). As is the case for most membrane proteins, they generally migrate faster in SDS-PAGE as an artifact of SDS-lipid micelle complex. The molecular weight of NET WT in total expression is observed as ∼54 kDa in SDS-PAGE, consistent with literature reports .max and Kd for NX at each of the mutants. Reactions containing membranes from hNET-transfected COS-7 cells (20 μg protein) and increasing concentrations of tritiated NX [(3H]-NX)] (0.125–50 nM) in assay buffer were established in clear round bottom 96-well plates. Each assay was performed in triplicate in a total reaction volume of 150 μl. The non-specific binding of [3H]-NX was determined in the presence of 200 μM NX. After 1 h incubation on ice, the membranes were harvested onto Whatman GF/B filtermats (PerkinElmer) pre-treated with 0.6% polyethylenimine using a Tomtec harvester. BetaPlate scintillant (PerkinElmer) was then applied and the filter-bound radioactivity detected using a Wallac MicroBeta (PerkinElmer). Specific [3H]-NX binding was calculated as the difference between the total and the non-specific binding. Each experiment was performed in triplicate and repeated 3–4 times. Total measured counts were always<10% of the counts added in all binding experiments.Saturation binding experiments were performed to determine the Bmax and the apparent Michaelis constant Km, defined as the extracellular NE concentration required for half-maximal transport velocity (Vmax). Cells were washed manually with 100 μl/well of hNET assay buffer -glucose, 1 mM ascorbic acid, pH 7.4). Uptake was initiated by the addition of 50 μl/well of hNET assay buffer containing increasing concentrations of [3H]-NE  from 31 nM–5 μM in triplicate. Non-specific uptake was measured using 200 μM of unlabeled NX. The uptake was abolished after 10 min incubation at 37°C by washing the cells twice with 100 μl/well of hNET assay buffer. Complete cell lysis was obtained by adding 50 μl of 0.1 M NaOH/well followed by 1 h incubation. Accumulated radioactive neurotransmitter was quantified using a Wallac MicroBeta counter (PerkinElmer Life Sciences). In each of the uptake experiment, the total measured counts were always <10% of the counts added.Adherent transfected cells were washed with 10 ml of phosphate-buffered saline (PBS) 24 h post transfection. The cells were then detached with 2 ml of Cell Dissociation Buffer Enzyme-Free PBS-based (Life technologies) followed by suspension of cells in growth medium. The cells were then added to 96-well plates at a density of 10,000 cells/well. Uptake assays were performed 24 h post plating to determine Vmax and Kd values were determined from saturation bindings curves, whereas Vmax and Km values were determined from saturation uptake curves. Data are presented as means ± SEM of results obtained from 3–15 separate experiments, each performed in triplicates. For multiple comparisons, one-way analysis of variance (ANOVA) was used with post hoc t-tests performed by Dunnett’s method using Prism (GraphPad Software). Values of P < 0.05 were considered significant. The calculations and statistical analysis on the Kd and Km data were performed on the log values.Curve fitting of saturation binding and transport kinetic data was performed by non-linear regression using the software package Prism (GraphPad Software). The BEvaluation of physical properties of homology models focuses on the problems resulting from protein structure outliers and steric clashes. Molprobity allows an all-atom contact analysis and quality check for identification of physical anomalies see , with th1+ through the carbonyl oxygen of A73 (TMH1) and S318 (TMH6), the hydroxyl oxygen of S318 (TMH6) and the side-chain carbonyl oxygen of D75 (TMH1), N78 (TMH1), and N350 (TMH8)  .Western blot analysis revealed that the A73G, A73V, A73L, A73R, A77L, A77R, N78A, V148A, N153A, G320A, V325A, N350A, S420A, G423A, and M424A mutants of hNET had similar expression levels compared to WT hNET when expressed in COS-7 cells. In contrast, the Y152A mutant did not express and the F72A, I156A, S318A, F323A, and F329A mutants showed reduced expression levels compared with WT hNET .max and Kd value for [3H]-NX at WT and mutant receptors were determined on cell membranes to establish expression levels and NX affinity, respectively. Specific binding  of [3H]-NX  significantly decreased for F72A (sixfold), A73R (fivefold), I156A (ninefold), G320A (14-fold), and F329A (12-fold) and increased for A73V (twofold), A77L (twofold), and S420A (twofold) compared to WT  of NX was reduced at A77R (sixfold), V148A (sevenfold), and N350A (12-fold) compared to WT  (Nisoxetine binding properties were determined for WT and mutant hNETs transiently expressed in COS-7 cells . The Bmaed to WT . These c n = 15) .3H]-NE  was saturable in COS-7 cells expressing hNET WT and all hNET mutants , A73G (twofold), A73L (21-fold), A73R (154-fold), A73V (twofold), A77L (98-fold), A77R (14-fold), N78A (sixfold), V148A (threefold), N153A (28-fold), I156A (20-fold), G320A (11-fold), F329A (22-fold), N350A (30-fold), and S420A (twofold) compared with hNET WT , A73V (threefold), A77L (threefold), N153A (twofold), G320A (twofold), G423A (fourfold), decreased for N350A (fourfold), and S420A (fourfold) compared to the WT (max (100%) or Km .Specific uptake of [ mutants . Signifi hNET WT . The Km o the WT . All othSubstrate binding to the S1 is considered a pivotal step in transporter function. This site lies deep within the transporter and overlaps with the S1 of orthosteric inhibitors . The S1 Transmembrane helice 1 is a highly conserved “signature” region of the SLC6 transporters. Our docking model revealed that four residues  in TMH1 of hNET contributed to the S1 pocket. Whereas F72 formed hydrophobic interactions with the aryl moiety of NE at 3.1 Å, the F72A mutant produced an equal drop in both the transport function and protein expression levels, indicating this residue contributed little to overall transport function but may play a structural role in NET. In support, F72V-NET was non-functional  and F72Y1+ ion (at 2.2 Å). The A73G- and A73V-hNET mutants had reduced  Km for NE and also significantly reduced transport, without affecting expression levels. Replacing alanine with the larger leucine and arginine (A73L- and A73R-hNET) had little effect on NE Km but almost completely abolished transport rate, suggesting these mutants do not contribute directly to NE binding but disrupt conformational changes required for transport. The A73G, A73V and A73L mutants had similar Bmax and Kd values for NX as WT hNET, whereas the A73R mutant had a reduced Bmax, likely linked to protein destabilization, that also influenced protein expression as showed by Western blot. The crystal structure of LeuT with leucine bound revealed that A22 in LeuT (corresponding to A73 in hNET) was involved in the octahedral co-ordination of Na+ via its carbonyl oxygen, supporting a structural role as well as providing hydrogen bonding to the amino group of leucine via its main chain carbonyl oxygen  and the Nal oxygen .m value), whereas A77R showed no change in NE potency  compared to WT. Bmax values with respect to WT were increased for A77L and similar for A77R. A77R showed a sixfold loss in the binding affinity of NX. These results suggest that like A73, A77R influence conformational changes relating to transport, perhaps through its role in maintaining the unwound loop connecting TMH1a-TMH1b. The crystal structure of LeuT with substrate leucine bound showed that G26 (corresponding to A77 in hNET) directly interacted through its amide nitrogen with the carboxyl group of leucine  and a hydrogen bond via the amide nitrogen of its main chain (at 3.0 Å). The A77G mutant has been tested earlier and shown to halve the transport rate . Both th leucine , similar leucine . This st leucine . Mutatin leucine . Thus, r1+ through its side-chain carbonyl oxygen. In agreement with our docking model, the alanine mutant of N78 (N78A) almost completely abolished NE transport without affecting the binding affinity of NE or NX. This result suggests that octahedral coordination of Na1+ might be critical for substrate transport. In addition, N78 could potentially be interacting with V244 in TMH7, a residue also important for efficient transport. We hypothesize that their interaction could play a role in transporter destabilization. Since N78 is conserved across the transporter SLC6 superfamily except in LeuT, which has a valine, the residue at this position is likely to play an important role in structure and function of the transporter.Our docking model of NE in hNET also showed that N78 contributed to the NE binding pocket by octahedral coordination of NaOur model also shows that V148, Y152, N153, and I156 in TMH3 contribute a hydrophobic surface to the NE binding pocket. A possible hydrophobic interaction of V148 with the aryl group of NE (3.3 Å) was confirmed experimentally as the alanine mutant V148A reduced transport rate ∼threefold and NE binding affinity ∼3.5-fold. The V148A mutant also reduced NX binding affinity sevenfold, indicating that V148 might form part of the NX binding pocket. V148 in hNET and its corresponding isoleucine residue in SERT are major determinants of SERT/NET selectivity for selective norepinephrine reuptake inhibitors (SNRIs) and serotonin reuptake inhibitors (SSRIs) . The corHydrogen bonding between Y152 and D75 in hNET also appears to be an important factor for maintaining a functional transporter and for NE binding. The Y152A hNET mutant abolished protein expression, whereas the previously studied Y152F mutant had only 6% transporter activity . The cormax and Kd values for NX were unaffected by this mutant, suggesting that transporter structure was unaffected. The N153S mutation has been shown to alter the hydrogen bonding properties and to reduce the transport activity to 19% of WT hNET and to decrease the binding affinity of NE, with no effect on norepinephrine reuptake inhibitor (NRI) binding  and S422 (hDAT), respectively . The G421+ via its side-chain carbonyl oxygen and is also anticipated to indirectly affect transporter function. Similar to what was observed for the G320A mutant, the N350A mutant abolished NE transport while increasing NE affinity, despite Bmax values for NX binding remaining at WT levels. However, affinity for NX was reduced 12-fold. Thus, we predict that the N350A mutant might stabilize conformations favoring substrate binding over antidepressant binding that prevents transporter cycling. N350 is conserved across the transporter SLC6 superfamily, with the corresponding N368 in SERT being highly sensitive to non-conservative replacements  or increased  NE affinity. 11 of 15 residues reduced NE transport, including several residues  that are conserved across monoamine transporters. Several mutations appear to restrict transporter cycling in a substrate  preferring conformation, which may help in identifying the structure of additional transporter conformational states and more specific transporter inhibitors.In conclusion, this study on hNET provides new insights into the structural features contributing to substrate specificity. Our docking studies showed that the catechol hydroxyl of NE did not form intermolecular interactions with the hNET, while the catechol benzene, catecholamine and β-hydroxyl of NE formed direct interactions with hNET. This suggests that the catechol hydroxyl part of NE is not essential for transport, explaining why hNET can transport both NE and DA. In contrast, hDAT does not transport NE, suggesting that the size of the catechol tail is key determinant of hDAT monoamine substrate specificity. We found that the residues involved in NaThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.PJ and RL participated in research design. PJ conducted experiments. PJ and LR performed the data analysis. PJ, LR, and RL wrote or contributed to the writing of the manuscript.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.


IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection. A recent study reported that anti-hemagglutinin (HA) head-targeting antibodies increase anti-viral functions such as hemagglutination inhibition (HI) and virus neutralization (NT), in addition to HA binding activity (reactivity) via IgA polymerization. However, the functional properties of anti-viral IgA antibodies with mechanisms of action distinct from those of anti-HA head-targeting antibodies remain elusive. Here, we characterized the functional properties of IgG, monomeric IgA, and polymeric IgA anti-HA stalk-binding clones F11 and FI6, and B12 , as well as Fab-deficient (ΔFab) IgA antibodies. We found that IgA polymerization impacts the functional properties of anti-HA stalk antibodies. Unlike anti-HA head antibodies, the anti-viral functions of anti-HA stalk antibodies were not simply enhanced by IgA polymerization. The data suggest that two modes of binding ) occur during interaction between anti-stalk HA IgA antibodies and HA. In situations where Fab paratope-mediated binding to the HA stalk exceeded IgA Fc glycan-mediated binding to HA RBS, IgA polymerization increased anti-viral functions. By contrast, when IgA Fc glycan-mediated binding to the HA RBS was dominant, anti-viral activity will fall upon IgA polymerization. In summary, the results suggest that coordination between these two independent binding modules determines whether IgA polymerization has a negative or positive effect on the anti-viral functions of anti-HA stalk IgA antibodies. Influenza is a highly contagious infectious disease caused by the influenza virus. The main targets of this virus in humans are epithelial cells lining the upper respiratory tract. Therefore, IgA antibodies, which are secreted onto the mucosal surface as secretory IgA antibodies (SIgAs), play an important role in preventing influenza virus infection –3. Curreet al. developed a method of generating polymeric SIgA in vitro, which involves co-transfecting mammalian cells with expression plasmids encoding the four components of SIgA: immunoglobulin heavy chain (HC), light chain (LC), joining chain (JC), and the secretory component (SC)  = 2.113 × 106) and an extremely low dissociation constant (kd (1/s) = below the measurement limit). An evident decrease in association (ka [1/Ms] = 8.541 × 103) and an increase in dissociation (kd (1/s) = 1.681 × 10–4) was observed in the presence of the T333K mutation (C1 virus). Although association of the G480D mutated virus (G6) decreased (ka [1/Ms] = 8.631 × 105), dissociation remained relatively low (kd (1/s) = 1.306 × 10–5) when compared with that for wild-type HA.Two escape mutant viruses of antibody clone F11 were obtained by serial passage of A/Narita/1/2009 (H1N1)pdm09 virus in the presence of high concentrations of F11 IgG1. (A) Escape mutant C1 exhibited higher escape efficiency than escape mutant G6. (B) Recombinant trimeric HA proteins harboring each mutation (T333K (mutant C) or G480D (mutant G)) were tested in a binding assay with F11 IgG1 to examine whether the mutations in the HA gene of escape mutant viruses affected the affinity of F11 for HA proteins. Binding of F11 IgG1 to HA harboring the T333K mutation (C1 virus) was notably lower than that to wild-type HA. By contrast, binding of F11 IgG1 to HA harboring the G480D mutation (G6 virus) was moderate when compared with that to wild-type HA. (C) The area under the reactivity curve (AUC) for F11 IgG1 in response to each recombinant HA . Data are expressed as the mean ± SD of three technical replicates. **(TIF)Click here for additional data file.S2 Figin silico characterization of HA trimer ectodomain structures by MD simulation. (B) Structure analysis of HA from wild-type NRT (obtained after 100 ns of MD simulation) showed that the T333K substitution occurred near the hydrophobic groove, a target of cross-clade neutralizing antibodies, whereas the G480D substitution arose at a site away from the groove. Three-dimensional location of escape mutations T333K and G480D (light green) in the HA trimer model of A/Narita/1/2009/H1N1. Purple spheres represent amino acid residues comprising hydrophobic grooves on the HA stalk. The groove of A/Narita/1/2009/H1N1 (GenBank accession number: ACR09396) comprises I389, V392, T393, and V396 in helix A, and W365 in the fusion peptide of the HA precursor. (C) Effect of the T333K and G480D mutations on the hydrophobic grooves. Hydrophobic surface patches on the stalk regions of HA trimers after 100 ns of MD simulation were identified using the protein patch analyzer in MOE and are shown in dark green. Red sticks denote side chains on residue 333 . Interestingly, the T333K substitution disrupted the physical continuity of the hydrophobic surface patches in the groove, possibly due to introduction of a large positively charged side chain (left and middle panels). By contrast, G480D mutant HA maintained the continuity of the hydrophobic patches (right panel). (D) Effect of the T333K and G480D mutations on the structural dynamics of HA trimers. RMSF values, which represent atomic fluctuations in the main chains of individual amino acids, were calculated using 15,000 snapshots obtained from MD simulations of 70–100 ns. HG indicates a region between positions 389 and 396, which contains residues I389, V392, T393, and V396 in the hydrophobic groove. The numbers on the horizontal axes indicate amino acid positions in the HA precursor of A/Narita/1/2009/H1N1. Notably, both the T333K and G480D substitutions induced marked changes in fluctuation profiles around the hydrophobic groove. Two independent MD simulations reproduced these results. Taken together, the results of the experimental and in silico studies suggest that the hydrophobic groove of the HA stalk plays a key role in interaction with clone F11. This is consistent with previous results showing that the hydrophobic groove plays a critical role in recognition of broadly neutralizing antibodies targeting the HA stalk. The data suggest that the human antibody clone F11 is an anti-HA stalk antibody with broadly neutralizing activity.To address the structural impact of these escape mutations in the HA stalk region, we conducted molecular dynamics (MD) simulation of the HA trimer ectodomains from the NRT, C1, and G6 viruses. (A) Flow chart showing (TIF)Click here for additional data file.S3 Fig(A–C) Chromatograms obtained by HPLC analysis of recombinant monomeric and polymeric IgA antibody clones B12 (A) and F11 (B), and of Fab-deficient (ΔFab) monomeric and polymeric IgA antibodies (C). Polymeric IgA antibody samples peaked at earlier time points than monomeric IgA antibody samples, indicating differences in molecular weight. (D) SDS-PAGE analysis of recombinant monomeric (M) and polymeric (P) F11, and of ΔFab IgA1 (A1) and IgA2m2 (A2m2) antibodies. All four components of secretory IgA antibodies (heavy chain [HC] (ΔFab HC in ΔFab IgA samples), light chain [LC], secretory component [SC], and the J chain [JC]) were observed. (E–I) Sedimentation velocity analytical ultracentrifugation (SV-AUC) was performed to determine the sedimentation coefficients  of FI6 IgG1 (E), IgA1 monomer (F), IgA2 monomer (G), IgA1 polymer (H), and IgA2 polymer (I) in solution. The main component  of IgG1 had a sedimentation coefficient typical of an antibody monomer (E). The s20,w of the main component of the IgA1 and IgA2 monomers was 6.6 S and 7.2 S, respectively, which is similar to that of the IgG1 used as a control for this study (F and G). For the IgA1 and IgA2 polymers, multiple oligomeric species were observed (H and I). The profiles of s20,w distribution did not change by diluting the samples from 1 mg/mL (black line) to 0.1 mg/mL (red line). This indicates that oligomerization of IgA is not an association-dissociation equilibrium, at least within this concentration range.(TIF)Click here for additional data file.S4 FigLC chromatograms of N-linked glycans released from human nasal IgA antibodies and recombinant (r) IgG/IgA antibodies. According to our test runs, peaks at later retention time points were more likely to contain terminally sialylated complex glycans; thus peaks observed at later time points were selected to estimate glycan composition. Peak patterns of IgA samples on LC chromatograms were similar, while those of IgG samples contained fewer peaks at later time points. Human nasal IgA samples generated the highest number of peaks at later time points. Peaks numbered 1 to 16 (red arrows) were estimated to contain terminally sialylated complex glycans. The estimated glycan compositions for each peak are shown in the left panels.(TIF)Click here for additional data file.S1 Raw imagesTop panel: Original image of the gel shown in (TIF)Click here for additional data file.


To test agreement, we used Lin’s concordance correlation coefficient and corresponding 95% confidence interval, Bland–Altman’s limits of agreement, and Bradley–Blackwood (BB) test. Agreement was high at 3 months , good at 12 months  but missing at 6 months of age . Overall, 18 infants had disparate results based on the cut-off point for vitamin D deficiency (25(OH)D < 50 nmol/L), particularly at three months, with seven (10%) infants deficient according to CIA but not LC–MS/MS, and four (6%) deficient by LC–MS/MS but not CIA. To our knowledge, this is the first study to show that the reported 25(OH)D concentration may be influenced by both age and assay type. Physicians and researchers should be aware of these pitfalls when measuring circulating 25(OH)D concentrations in infants and when developing treatment plans based on measured vitamin D status.Hypovitaminosis D is prevalent worldwide; however, analytical bias in the measurement of circulating 25-hydroxyvitamin D (25(OH)D) concentrations may affect clinical treatment decisions and research. We performed parallel plasma 25(OH)D analyses using the Abbott Architect i2000 chemiluminescent immunoassay (CIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for paired samples from the same infants at 3 ( Vitamin D deficiency is a significant global concern due to both its high prevalence in diverse populations and its multisystem health implications ,2,3,4,5.Many of these multisystem effects may be influenced by the impact of vitamin D on the immune function . Increas3 plus 25(OH)D2) [3), particularly in infants aged ≤12 months [3 may be incorrectly interpreted as 25(OH)D by some assays. Furthermore, the downstream hydroxylated C3-1,25(OH)2D is thought to have considerably lower biological activity than the active form of vitamin D, 1,25-dihydroxyvitamin D. Inadvertently including C3-epi-25(OH)D3 may result in an overestimation of circulating 25(OH)D concentrations in infants, leading to under-prescription of supplements and undue toxicity concerns [Thresholds for vitamin D deficiency are based on total 25(OH)D concentrations (25(OH)D5(OH)D2) ,13. Howe5(OH)D2) ,14. The 2 months ,16. Due concerns ,16.3 concentrations independently of 25(OH)D2 and C3-epi-25(OH)D3 [A liquid chromatography–tandem mass spectrometry (LC–MS/MS) method that is certified to the reference measurement procedures (RMPs) developed by the National Institute of Standards and Technology, Ghent University, and the US Centers for Disease Control and Prevention ,18 is co25(OH)D3 . However25(OH)D3 . This is25(OH)D3 .Despite the introduction of monitoring programs  , VitaminLimited data on assay variability are available in infants ,26. An AThis is an observational study comparing parallel analyses by CIA and LC–MS/MS of identical paired samples for plasma 25(OH)D concentrations in the same cohort of infants at 3, 6, and 12 months of age. The samples were collected between March 2012 and August 2016 as part of a randomized controlled trial conducted in Perth, Australia , which wPeripheral blood was collected by venipuncture and processed for plasma and mononuclear cells. Ref.  Two diff3 epimer was 1.3%, and that between 25(OH) vitamin D and 25(OH)D2 epimer was 0.8%. The Abbott Architect i2000 is accredited by the National Association of Testing Authorities for measurement of 25(OH)D, and the recently improved version has been shown to have reduced variability in comparison to LC–MS/MS [We used a delayed one-step chemiluminescent microparticle immunoassay (CIA) on an automated Abbott Architect i2000 . The calibrators were standardized against Standard Reference Material 2-972 from the National Institute of Standards and Technology . The anaLC–MS/MS .2, 25(OH)D3 and C3-epimer concentrations were measured; 25(OH)D2 concentrations were consistently <3 nmol/L and were therefore not reported [The concentrations of 25(OH)D were also analysed by the LC–MS/MS method certified by the VDSP . This mereported . As Abbop < 0.05. Concordance was implied if the value for the correlation between difference and mean was near zero and the Bradley–Blackwood coefficient (F) was not statistically significant. Vitamin D deficiency was defined as 25(OH)D concentrations < 50 nmol/L, the preferred cut-off point used in Australia [Statistical analysis was conducted using Stata v14 . As measures of agreement, we used Lin’s concordance correlation coefficient and corresponding 95% confidence interval and Bland–Altman’s limits of agreement method. Bradley–Blackwood test, a test of equality of means and variances of both variables, was also performed. All statistical tests were two-tailed. Statistical significance was set at ustralia ,35. We custralia ,37.Of the 120 participants, parallel plasma 25(OH)D measurements using CIA and LC–MS/MS were available for paired samples of 69 infants at 3 months, 79 infants at 6 months, and 73 infants at 12 months of age .2 and D3 metabolites. Using LC–MS/MS, we were able to differentiate D2 and D3 metabolites, and this analysis consistently revealed 25(OH)D2 concentrations of <3 nmol/L for each included sample.The Abbott CIA reported the sum of Dp = 0.443) (p < 0.001) (p = 0.097)   but not < 0.001) . There w= 0.097) .For each assay, we also determined the proportion of samples identified as below the cut-off point for vitamin D deficiency (25(OH)D < 50 nmol/L). Seven (10%) of the three-month-old infants were defined as vitamin D-deficient by CIA but not LC–MS/MS, and four (6%) were defined as deficient by LC–MS/MS but not CIA . Among tThis study compared the two major 25(OH)D assays currently in use (immunoassay and LC–MS/MS) for analysing plasma 25(OH)D concentrations in the same cohort of 3-, 6-, and 12-month-old infants. We found that inter-assay variation differed by age. Overall, there was agreement at 3 and 12 months, but not at 6 months of age. On an individual basis, measurements around the cut-off point for vitamin D deficiency (25(OH)D < 50 nmol/L) differed between assays. At three months of age, 10% of the infants were defined as vitamin D-deficient by CIA but not LC–MS/MS, and 6% were defined as deficient by LC–MS/MS but not CIA. Hence, such assay variability may lead to therapeutic consequences for younger infants, including errors in the determination of the need and dose of vitamin D supplementation to promote bone health.Uniquely, we compared CIA and LC–MS/MS longitudinally in the first year of life and noticed an age difference in assay variability which is supported by a European study in older children and adults , suggest3 and the C3-epimer is known to be only minimal (1.3%). Hence, detection of the C3-epimer would not explain the missing agreement between the two assays at six months of age. Potentially, it could be argued that the C3-epimer concentrations may have affected the outcome of the comparison between the two assays. However, in our study, C3-epimer concentrations were very similar at three and six months of age, although our results showed agreement between methods at three months of age but not at six months of age. Furthermore, looking at CIA, cross reactivity between 25(OH)Dn = 10) South Australian study on neonates [While our results are consistent with those of the previous small  with a high-pressure liquid chromatography (HPLC) method for measuring 25(OH)D concentrations . They foStudies in adults also demonstrate inter-assay variability ,24,38,40Since accurate and reliable measures are essential for clinical indications for vitamin D supplementation, assay variability may have an influence on clinical decisions. Correct clinical decisions, while important at all ages, are particularly important in early childhood ,44, a liAssay variability may also impact on research, since the use of different assays hinders pooling of results. A few randomized controlled trials have assessed the efficacy of daily drops of 400 IU vitamin D in the first year of life. However, different assays were used to quantify 25(OH)D: CIA ,45, radiAlthough efforts have been made to standardize 25(OH)D measurements in research and clinical settings ,21,22,42A strength of the present study is the analysis of sample pairs in the same cohort of infants at 3, 6 and 12 months of age. However, our study was a single-centre study with a limited number of analysed samples; therefore, results may not be generalizable. The inherent limitations of vitamin D assays must also be considered when comparing variability, including variations between and within methods carried out in different laboratories, by different operators, or using different reagent kit batches.We have shown that age in infancy influences the variability between commonly used assays for measuring 25(OH)D concentrations. Physicians, researchers, laboratory technicians and authorities need to be aware of the current limitations when interpreting and comparing vitamin D values derived from different assays. Accurate measurement of circulating 25(OH)D concentrations is essential for indicating the need for vitamin D supplementation, which is particularly important in pregnancy, lactation and infancy, when optimal bone growth is of paramount importance. Reliable measurement is also crucial for research and public health initiatives, particularly for estimating the prevalence of vitamin D deficiency and for following participants longitudinally.


Niemann-Pick C1-Like 1 (NPC1L1) is a cholesterol importer and target of ezetimibe, a cholesterol absorption inhibitor used clinically for dyslipidemia. Recent studies demonstrated that NPC1L1 regulates the intestinal absorption of several fat-soluble nutrients, in addition to cholesterol. The study was conducted to reveal new physiological roles of NPC1L1 by identifying novel dietary substrate(s). Very low-density lipoprotein and low-density lipoprotein (VLDL/LDL) are increased in Western diet (WD)-fed mice in an NPC1L1-dependent manner, so we comprehensively analyzed the NPC1L1-dependent VLDL/LDL components. Apolipoprotein M (apoM), a binding protein of sphingosine-1-phosphate (S1P: a lipid mediator), and S1P were NPC1L1-dependently increased in VLDL/LDL by WD feeding. S1P is metabolized from sphingomyelin (SM) and SM is abundant in WD, so we focused on intestinal SM absorption. In vivo studies with Npc1l1 knockout mice and in vitro studies with NPC1L1-overexpressing cells revealed that SM is a physiological substrate of NPC1L1. These results suggest a scenario in which dietary SM is absorbed by NPC1L1 in the intestine, followed by SM conversion to S1P and, after several steps, S1P is exported into the blood as the apoM-bound form in VLDL/LDL. Our findings provide insight into the functions of NPC1L1 for a better understanding of sphingolipids and S1P homeostasis. Sphingolipids constitute a class of lipids that are essential components of membranes in eukaryotes. In addition to their primary roles in membrane formation, some bioactive sphingolipids and their metabolites  and sphingosine-1-phosphate (S1P)) have attracted attention as important regulators of multiple cellular behaviors and responses, including cell proliferation, cell death, and immune responses ,3. Howev1, in addition to cholesterol , where P was the probability that the observed match was a random event, by using Proteome Discoverer version 1.3.Shotgun-proteomics analyses were conducted using an EASY-nLCeviously . BrieflyTM Tandem Quadrupole Mass Spectrometer coupled to an ACQUITY Ultra Performance LC (UPLC) System with an integral autoinjector . The XEVO spectrometer was run in electrospray ionization-MS/MS multiple reaction monitoring mode at a source temperature of 120 °C and a desolvation temperature of 350 °C. The sample temperature was maintained at 4 °C and the column temperature was maintained at 50 °C. The mobile phases were 0.1% formic acid solution (solvent A) and liquid chromatography-grade acetonitrile  (solvent B). The UPLC and mass spectrometer conditions are shown in Fractionated plasma samples were mixed with an internal standard solution (100 ng/mL of pioglitazone), followed by deproteinization with 4 volumes of methanol and 4 volumes of acetonitrile. After centrifugation at 15,000 rpm for 5 min, deproteinized supernatants were evaporated using a Speed-Vac concentrator , and the dried samples were dissolved in methanol. To quantify the S1P concentrations in each sample, LC/MS/MS multiple reaction monitoring analyses were conducted on a XEVOData analyses were performed using MassLynxNT software version 4.1 (Waters).3H] SM: Sphingomyelin (bovine) [choline methyl-3H] . The solvent was evaporated, and 19 mM sodium taurocholate (dissolved in PBS) was added to obtain the required lipid concentrations. The mixture was sonicated three times for 3 min using an ultrasonic homogenizer.Emulsions were prepared as described previously , with mi3H] SM-containing emulsion (5 mL/kg body weight) was delivered into the small intestine via the duodenal cannula. Two hours after emulsion loading, the mice were sacrificed, and the plasma and liver were isolated to quantify the level of absorbed [3H] SM. The radioactivity of each specimen was measured using a liquid scintillation counter.In vivo acute absorption studies were performed as described previously , with mi2.NPC1L1 overexpressing Caco-2 cells and control Caco-2 cells were generated in our previous studies by stable transfection of a pcDNA3.1(+) vector carrying human NPC1L1 cDNA with hemagglutinin (HA) tag (YPYDVPDYA) sequences at the 3′-end and an empty vector [pcDNA3.1(+)], respectively ,7,14. AlCaco-2 cells were lysed with radioimmunoprecipitation assay buffer . Total extracted cellular proteins (40 μg) were diluted with 2× SDS loading buffer and subjected to Western blot analysis, as described previously . An SDS–3H] SM diluted in ethanol (0.04 μCi/mL) were mixed with or without (for mock control) ezetimibe diluted in methanol (40 μM) and then evaporated to dryness with mild heating under N2 gas. Transport buffer  was added to prepare the medium for uptake experiments. The [3H] SM-containing medium was thoroughly vortexed and stirred at 37 °C for 2–3 h.Cholesterol diluted in ethanol (1 μM), phosphatidylcholine diluted in methanol (50 μM), sodium taurocholate diluted in 96% ethanol (2 mM), and  SM-containing medium was added, and the cells were incubated for 3 h. The cells were then washed with ice-cold transport buffer and disrupted with 0.2 N NaOH overnight. [3H] SM in the cell lysate was measured with a liquid scintillation counter to determine its cellular uptake. For normalization, the protein concentration of each well was determined using a BCA protein assay kit (Thermo Fisher Scientific).In vitro uptake assays were performed as described previously ,9,14,15,The project identification codes are P14-111 (approved date: 16 October 2014) and P19-052 (approved date: 6 September 2019).WT mice were observed) and long-term (20 weeks: atherosclerotic plaques in Npc1l1WT mice were cleary observed) )-labeled SM. The amount of SM absorbed in the plasma and liver was 70% (plasma), 56% (liver), and 57% (plasma + liver) lower in Npc1l1KO mice than in Npc1l1WT mice  have lower diet-induced VLDL/LDL-apoM-S1P, indicate that CETP and NPC1L1 play key roles in regulating VLDL/LDL-apoM-S1P in humans, particularly in patients with dyslipidemia.In this study, we first demonstrated that NPC1L1 regulates S1P distribution to VLDL/LDL particles in mice fed a WD. S1P is thought to be bound with apoM or albumin in the blood, and most apoM-bound S1P is distributed to HDL particles in healthy mice . HoweverThe physiological functions of S1P vary depending on its mode of existence in the blood. Indeed, unlike albumin-bound S1P, apoM-bound S1P on HDL (HDL-apoM-S1P) has been reported to transduce anti-inflammatory signals by activating the S1P receptor 1 in vascular endothelial cells, resulting in downregulation of proinflammatory adhesion proteins such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 . In addi3H]-choline-labeled SM, we were unable to discriminate the absorption of intact SM from that of SM-derived choline, which is enzymatically produced from SM in the intestinal lumen; current understanding of SM digestion and absorption is that luminal sphingomyelin is hydrolyzed in the middle-to-distal small intestine by alkaline sphingomyelinase in the intestinal lumen, to yield ceramide and phosphocholine [Another important finding of the present study is that NPC1L1 takes up SM, and intestinal absorption of dietary SM was found to be regulated by NPC1L1 in an ezetimibe-sensitive manner . Our resocholine . Phosphoocholine —it is poKO mice and ezetimibe-administered Npc1l1WT mice (Second, detailed molecular mechanisms of NPC1L1-mediated SM uptake remain unknown. Regarding the NPC1L1-mediated uptake of cholesterol, it has been reported that N-terminal domain of NPC1L1 binds cholesterol and promotes the formation of cholesterol-enriched microdomain in the plasma membrane to facilitate cholesterol transport ,25. In a1WT mice  is consiIn conclusion, our results indicate that NPC1L1, a cholesterol importer, is involved in the uptake of dietary SM and controls diet-induced VLDL/LDL-apoM-S1P levels in the blood . This is


THOC1 as the probable cause of the late-onset, progressive, non-syndromic hearing loss in a large family with autosomal dominant inheritance. Thoc1, a member of the conserved multisubunit THO/TREX ribonucleoprotein complex, is highly expressed in mouse and zebrafish hair cells. The thoc1 knockout (thoc1 mutant) zebrafish generated by gRNA-Cas9 system lacks the C-startle response, indicative of the hearing dysfunction. Both Thoc1 mutant and knockdown zebrafish have greatly reduced hair cell numbers, while the latter can be rescued by embryonic microinjection of human wild-type THOC1 mRNA but to significantly lesser degree by the c.547C>G mutant mRNA. The Thoc1 deficiency resulted in marked apoptosis in zebrafish hair cells. Consistently, transcriptome sequencing of the mutants showed significantly increased gene expression in the p53-associated signaling pathway. Depletion of p53 or applying the p53 inhibitor Pifithrin-α significantly rescued the hair cell loss in the Thoc1 knockdown zebrafish. Our results suggested that THOC1 deficiency lead to late-onset, progressive hearing loss through p53-mediated hair cell apoptosis. This is to our knowledge the first human disease associated with THOC1 mutations and may shed light on the molecular mechanism underlying the age-related hearing loss.Apoptosis of cochlear hair cells is a key step towards age-related hearing loss. Although numerous genes have been implicated in the genetic causes of late-onset, progressive hearing loss, few show direct links to the proapoptotic process. By genome-wide linkage analysis and whole exome sequencing, we identified a heterozygous p.L183V variant in  THOC1 as the probable cause of the hearing loss in a large family with autosomal dominant inheritance. In addition, we showed THOC1 was expressed in mouse and zebrafish hair cells. Furthermore, we revealed that thoc1 deficiency caused the reduction of hair cell numbers in zebrafish and the hypomorphic Thoc1 induced hair cell apoptosis. These defects could be attenuated by the inhibition of p53.We identified a variant in  Age-related hearing loss (ARHL) affects over 40% of the population older than 65 years . Based ohttp://hereditaryhearingloss.org/). In addition, a number of genes and loci, including GRM7 and IQGAP2 for the former, have been implicated in ARHL by large-scale genome-wide association studies .Members of Family SH was recruited through the Affiliated Hospital of Nantong University in Nantong, Jiangsu Province, China. A total of 9 affected and 18 unaffected members participated in the present study  and based on genotypes of 6301 Tag SNPs with an averaged 0.5cM resolution. Logarithm of the odds (LOD) scores were calculated by the parametric linkage analysis package Merlin v. 1.1.23. The inheritance model was set to dominant with full penetrance. The disease allele frequency was set to 0.0001.http://www.mutationtaster.org), PROVEAN and SIFT .Whole-exome sequencing was performed in four affected  and two unaffected (III-16 and IV-6) members (marked with triangles in Immunofluorescence staining of THOC1 was performed in cross-section and whole mount samples of mouse inner ear as previously described . All proTg(pou4f3:gap43-GFP) and Tg(cldnb:lynGFP) were used as previously described [The zebrafish embryos and adults were maintained in zebrafish Center of Nantong University under standard conditions in accordance with our previous procedures , 36. Theescribed . Sound-ebax: 5’-AGAGGGTGAAACAGACCAGC-3’ and 5’-GCTGAACAAGAAAGGGCACAG-3’; for caspase3: 5’-TGGCACTGACGTAGATGCAG-3’ and 5’- GAAAAACACCCCCTCATCGC-3’; for caspase9: 5’-ACTAAATGACCGCAAGGGCT-3’ and 5’-TTGCCTCAGTGCCATGTGAA-3’; for p53: 5’-GCAAAAACTTGCCCCGTTCA-3’ and 5’-GCTGATTGCCCTCCACTCTT-3’.Total RNA was extracted from zebrafish embryos by TRIzol reagent according to the manufacturer’s instructions . Genomic contaminations were removed by DNaseI, and then 2 μg total RNA was reversely transcribed using a reversed first strand cDNA synthesis kit  and stored at −20°C. The sequences of PCR primers, used for validating the Splice-blocking Morpholino, were: 5’-TCCGTCTCACTTCGACTTCA-3’ and 5’-TCCCAGCAGAGTAAAATGTGT-3’. The Realtime PCR analysis was performed according to the procedure described in our previous work . The prithoc1 F1 5’-TGGAATCTGAACCCCGACAA-3’ and thoc1 R1 5’-TGTCCGACTCGATCACTCTG-3’. Digoxigenin-labeled sense and antisense probes were synthesized using linearized pGEM-Teasy vector subcloned with this thoc1 fragment by in vitro transcription with DIG-RNA labeling Kit . Zebrafish embryos and larvae were collected and fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for one night. The fixed samples were then dehydrated through a series of increasing concentrations of methanol and stored at −20°C in 100% methanol eventually. Whole mount in situ hybridization was subsequently performed as described in the previous study [A 498 bp cDNA fragment of Thoc1 was amplified from the cDNA library that established from wild type (WT) AB embryos using the specific primers of us study , 38.Cas9 mRNA was obtained by in vitro transcription with the linearized plasmid pXT7-Cas9 according to the procedure previously described. The sgRNAs were transcribed from the DNA templates that amplified by PCR with a pT7 plasmid as the template, a specific forward primer and a universal reverse primer. The gRNA sequence is listed in the following: 5’-TAATACGACTCACTATAGGTGATTAAAACCGGAGAGGGTTTTAGAGCTAGAAATAGC-3’. Cas9 mRNAs were synthesized in vitro using the linearized constructs as templates with SP6/T7 mMESSAGE mMACHINE Kit , purified with RNeasy Mini Kit , and dissolved in RNase free Ultrapure water . The sgRNAs were synthesized by the MAXIscript T7 Kit , and were purified with RNeasy Mini Kit , and dissolved in RNase free Ultrapure water . Zebrafish lines were naturally mated to obtain embryos for microinjection. One to two-cell stage zebrafish embryos was injected with 2–3 nl of a solution containing 250 ng/μl Cas9 mRNA and 15 ng/μl sgRNA. At 24 h post fertilization (hpf), zebrafish embryos were randomly sampled for genomic DNA extraction according to the previous methods to determine the indel mutations by sequencing.Thoc1 splice-blocking Morpholino  sequence was 5′- AGTAAGCTGTGGACTCACTATCTGC -3′. The sequence of a standard control MO oligo was 5′-CCTCTTACCTCAGTTACAATTTATA-3′. The MOs were diluted to 0.3 mM with RNase-free water and injected into the yolk of one to two-cell stage embryos and then raised in E3 medium at 28.5°C. The cDNAs containing the open reading frame of the target genes were cloned into PCS2+ vector respectively and then was transcribed in vitro using the mMESSAGE mMACHIN Kit  after the recombinant plasmids linearized with NotI Restriction Enzyme , and then the capped mRNAs were purified by RNeasy Mini Kit . 2 nl target genes mRNA were injected at 50 ng/μl into 1/2-cell stage embryos.To visualize and image the hair cells in lateral line neuromasts, the vital dye FM1-43FX  was applied at a concentration of 3 μM to live larvae for 45 s in the dark. After quickly rinsing three times with fresh water, the larvae were anesthetized in 0.02% MS-222 and fixed with 4% PFA in PBS for 2 h at room temperature or overnight at 4°C. 5 μM Pifithrin-α  were diluted in the E3 solution. The embryos were treated from 3 dpf.In Situ Cell Death Detection Kit  following the directions supplied by the manufacturer.For TUNEL  assays, larvae were incubated in 0.1 M glycine/PBS solution for 10 min and then rinsed with PBT-2 three times for 10 minutes each. The larvae were then processed using the Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen). RNA samples were quantified and qualified by Agilent 2100 Bioanalyzer . Next generation sequencing library preparations were constructed according to the manufacturer’s protocol . The libraries were sequenced using Illumina HiSeq instrument according to manufacturer’s instructions . The sequences were processed and analyzed by GENEWIZ. Differential expression analysis was carried out using the DESeq Bioconductor package. After adjusted by Benjamini and Hochberg’s approach for controlling the false discovery rate, P-value of genes were set <0.05 to detect differential expressed ones.in situ hybridization, Photographs were taken using an Olympus stereomicroscope MVX10. All images of THOC1 immunostaining in mouse were imaged using the Leica SP5 confocal microscope. Statistical analysis was performed using GraphPad Prism software. T-test and one-way analysis of variance (ANOVA) were used, and P < 0.05 were considered statistically significant.Zebrafish embryos were anesthetized with E3/0.16 mg/mL tricaine/1% 1-phenyl-2-thiourea  and embedded in 0.8% low melt agarose, and then were examined with a Leica TCS-SP5 LSM confocal imaging system. For the results of S1 FigHearing thresholds were averages of both ears. All affected members exhibited symmetric audiometric configuration.(PDF)Click here for additional data file.S2 FigP>0.05, t-test) differential expression in the affected individual in comparison with that of the internal control gene (GAPDH).Relative expression level is shown as the ratios of that of the affected individual III-10 over the unaffected individual IV-6. None of the 11 genes show significant ((PDF)Click here for additional data file.S3 Fig(a) Primer design that amplifies multiple exons (4–10) containing and flanking the c.547C>G mutation (red dash) in exon 7. (b) No differential mRNA expression or alternatively spliced transcript can be detected in the affected individual III-10. (c) Sequencing results of the amplicons.(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 FigTHOC1 expression in several cell lines.(a) Western blot analysis of THOC1 expression in 293t cells and THOC1 knockout 293t cells. (b) Realtime PCR analysis of (PDF)Click here for additional data file.S6 FigConfocal microscopic imaging analysis of THOC1 antibody staining in P0 mouse. Blue: DAPI staining of the cell nuclei. Red: Myosin 7a staining marking hair cells. Green: THOC1 antibody staining. Bars, 40 μm.(PDF)Click here for additional data file.S7 Fig(a) The targeting sites of gRNAs. (b) Mutation pattern of gRNA1/gRNA2 and cas9 mRNA coinjected embryos. Numbers in the brackets show the number of nucleotides were deleted (-) or inserted (+). Inserted nucleotide is in red. WT, wild type.(PDF)Click here for additional data file.S8 FigTg(cldnb:lynGFP) zebrafish embryos at 48 hpf. (b) The neuromasts detected by whole mount in situ hybridization analysis of eya1 at 48 hpf. (c) The fluorescence microscopic imaging analysis of control and thoc1 mutants Tg(cldnb:lynGFP) zebrafish embryos at 3 dpf. (d) The statistical analysis of the number of neuromasts at each side of the control (n = 12) and thoc1 mutant (n = 24) embryo trunk at 48 hpf. t-test, ****P < 0.0001. (e) The statistical analysis of the number of neuromasts at each side of the control (n = 12) and thoc1 mutant (n = 23) embryo trunk at 3 dpf. t-test, ****P < 0.0001.(a) The fluorescence microscopic imaging analysis of control and thoc1 mutants (PDF)Click here for additional data file.S9 Figthoc1 mutants Tg(pou4f3:gap43-GFP) at 3 dpf. (b) Statistical analysis of the hair cells in otic vesicle of control and thoc1 mutants. t-test, ****, p<0.0001.(a) Confocal microscopic imaging analysis the hair cells in otic vesicle of control and (PDF)Click here for additional data file.S10 Fig(PDF)Click here for additional data file.S11 Fig(PDF)Click here for additional data file.S12 Fig(PDF)Click here for additional data file.S13 Figp53 knockout (KO), thoc1 morphants, and p53 KO+ thoc1-MO embryos stained with FM1-43FX at 3 dpf.  Statistical analysis of the hair cell clusters in trunk and head of control, p53 knockout (KO), thoc1 morphants, and p53 KO+ thoc1-MO embryos. One-way ANOVA, ****P<0.0001, **P<0.01. (d) Confocal imaging analysis of the hair cells in control, p53 knockout (KO), thoc1 morphants, and p53 KO+ thoc1-MO embryos.(a) The fluorescence microscopic imaging analysis of control, (PDF)Click here for additional data file.S14 FigArrowheads indicate hair cell clusters.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Data(PDF)Click here for additional data file.


IDH1) R132H, BRAF V600E, and the TERT promoter in 83 diffuse spinal cord astrocytic tumors. Among these samples, thirty-five patients had the H3 K27M mutation; this mutant could be observed in histological grade II (40%), III (40%), and IV (20%) astrocytomas. IDH1 mutations were absent in 58 of 58 cases tested. The BRAF V600E mutation (7/57) was only observed in H3-wildtype astrocytomas, and was associated with a better prognosis in all histological grade II/III astrocytomas. TERT promoter mutations were observed in both H3 K27M-mutant (4/25) and -wildtype (9/33) astrocytomas, and were associated with a poor prognosis in H3-wildtype histological grade II/III astrocytomas. In the 2016 WHO classification of CNS tumors, H3 K27M-mutant diffuse midline gliomas, including spinal cord astrocytomas, are categorized as WHO grade IV. Here, we noticed that the median overall survival of histological grade II/III H3 K27M-mutant cases (n = 28) was significantly longer than that of either the total histological grade IV cases (n = 12) or the H3 K27M-mutant histological grade IV cases (n = 7). We also directly compared H3 K27M-mutant astrocytomas to H3-wildtype astrocytomas of the same histological grade. In histological grade II astrocytomas, compared to H3-wildtype cases (n = 37), H3 K27M-mutant patients (n = 14) had showed a significantly higher Ki-67-positive rate and poorer survival rate. However, no significant differences in these parameters were observed in histological grade III and IV astrocytoma patients. In conclusion, these findings indicate that spinal cord astrocytomas are considerably different from hemispheric and brainstem astrocytomas in terms of their molecular profiles, and that the histological grade cannot be ignored when assessing the prognosis of H3 K27M-mutant spinal cord astrocytomas.Due to the rare incidence of spinal cord astrocytomas, their molecular features remain unclear. Here, we characterized the landscapes of mutations in H3 K27M, isocitrate dehydrogenase 1 ( Currently, accurate molecular pathological information is vital for the precise diagnosis and clinical management of gliomas in the brain; their key molecular features, including isocitrate dehydrogenase (IDH) mutations, 1p/19q co-deletion, and the H3 K27M mutation, are included in the World Health Organization (WHO) 2016 classification of tumors of the central nervous system , 25. AltAccording to the 2016 WHO classification, diffuse midline gliomas with an H3 K27M mutation in either H3F3A or HISTIH3B/C are considered a novel entity known as “diffuse midline glioma, H3 K27M-mutant” . This noWhether the prognostic value of the H3 K27M mutation is influenced by tumor location remains uncertain. The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy—Not Official WHO—have clarified the diagnostic criteria for “Diffuse midline glioma, H3 K27M-mutant,” and suggested that this term should be reserved for tumors that are diffuse , midline  gliomas with the H3 K27M mutation, and should not be applied to other tumors with the H3 K27M mutation . A previIDH1 R132H mutation, BRAF V600E mutation, TERT promoter mutation, and their prognostic value in 58 cases (57 for BRAF V600E mutation). Importantly, we also directly compared the clinicopathological features and prognoses of H3 K27M-mutant and H3-wildtype astrocytomas in each of the grade II, III, and IV samples.Here, we retrospectively analyzed the clinical and basic molecular pathological features of 83 patients with spinal cord astrocytomas diagnosed between 2011 and 2018 in the Beijing Tiantan Hospital. In addition to clinical characteristics such as age, gender, anatomic tumor location, and extent of tumor resection, the H3 K27M-mutant status of all patients was identified. We also analyzed the distributions of We retrospectively collected the data of 83 patients with diffuse astrocytic tumors in the spinal cord between 2011 and 2018 in the Department of Neurosurgery at Beijing Tiantan Hospital, China  after PCR amplification in 66 cases where sufficient DNA could be extracted from FFPE samples ; the PCR and sequencing primers used are listed in Supplementary Table MGMT promoter methylation was also assessed by PSQ with the PyroMark Q24 MGMT kit on a PyroMarker Q24 instrument (Qiagen), as previously described  = 1.84, 95% confidence interval [CI]: 1.13–3.01, P = 0.014] and H3 K27M-mutant status  were significantly associated with OS in multivariate Cox regression analysis.In all diffuse spinal cord astrocytic tumors, we observed that the median OS of H3 K27M-mutant cases was significantly shorter P < 0.000 than thaP < 0.0001) and integrated diagnosis of 2016 WHO classification (P < 0.0001) successfully stratified the OS of spinal cord gliomas. However, we noticed that the median OS (20.53 months) of patients with 2016 WHO grade IV astrocytomas, including histological grade IV astrocytomas and H3 K27M-mutant histological grade II/III astrocytomas, was longer than that (10.42 months) of patients with only histological grade IV astrocytomas   Fig. f. We spen = 14) showed a lower ratio of GTR , a higher ratio of Ki-67-positive samples , and a lower survival rate (P < 0.0001) only in histological grade II astrocytomas, but not in histological grade III and histological grade IV astrocytomas. There were no significant differences in all characteristics (except for BRAF V600E mutation) and prognosis between H3-wildtype and H3 K27M-mutant histological grade III gliomas. The age at diagnosis of H3 K27M-mutant histological grade IV cases was significantly younger  than that of H3-wildtype histological grade IV cases.The above findings demonstrated that the histological grade cannot be ignored in H3 K27M-mutant astrocytomas, and that the impact of H3 K27M mutation on spinal cord astrocytomas may also differ in astrocytomas with different histological grades. Thus, we also comprehensively compared the clinicopathological features and prognoses of H3 K27M-mutant and H3-wildtype gliomas in each of the grade II, III, and IV samples  of spinal cord astrocytoma cases with different histological grades. Moreover, we noticed that the median age of H3 K27M-mutant spinal cord astrocytomas was 35 years (range: 9–52 years), and that the median OS of H3 K27M-mutant spinal cord astrocytomas was 20.7 months; this is consistent with previous reports, but significantly different from that of brainstem astrocytomas, which mainly occurs in adolescents and shows a median OS of less than 12 months [Although ther low , 35. No ocytomas , 31. Simd glioma . Here, w2 months , 28, 33.TERT promoter mutations are associated with a poor survival rate in IDH-wildtype lower-grade brain gliomas [TERT promoter mutation in spinal cord glioma was suggested in three cases [TERT promoter mutations was 22.4% (13/58); additionally, this mutation was found mainly in H3-wildtype tumors (9/13), and was associated with a poor prognosis for grade II/III spinal cord gliomas. The BRAF V600E mutation, KIAA1549-BRAF fusion, FGFR alterations, and MYB or MYBL1 rearrangements are key prognostic and therapeutic markers for diffuse brain gliomas with wildtype IDH and H3 in children or adolescents [BRAF V600E only occurred in H3-wildtype grade II/III spinal cord astrocytomas, and was associated with a good prognosis. These findings suggest that the BRAF V600E and TERT promoter mutant status should also be included in the molecular testing of spinal cord gliomas, especially for histological grade II or III gliomas.It has been suggested that  gliomas , 29, 36.lescents , 15, 30.Although diffuse midline gliomas with the H3 K27M mutation have been suggested to correspond to a grade IV classification, the impact of the H3 K27M mutation on spinal cord astrocytomas may be different from its impact on brainstem glioma , 33, 38.P < 0.0001), but not in histological grade III (P = 0.1297) and IV (P = 0.7495) tumors. A similar result has been reported in a study that pooled midline gliomas from different anatomic locations [In present study, we analyzed 14, 14, and 7 cases of H3 K27M-mutant spinal cord glioma with histological grade II, III and IV, respectively. Thus, we could directly compare the H3 K27M-mutant astrocytomas to H3-wildtype astrocytomas of the same histological grade. We confirmed that the impact of the H3 K27M mutation on spinal cord glioma prognosis was influenced by the histological grade. The H3 K27M mutation was associated with a poor survival rate only in histological grade II tumors (ocations . MoreoveThe limitations of this study include its retrospective design, that H3 K27M-mutant status was only determined by immunohistochemistry, and that only the basic molecular features of the tumors were analyzed due to a lack of available samples for adequate analyses. Future studies with large-scale molecular analyses are warranted to confirm the specificity of spinal cord astrocytomas, and to identify novel molecular markers for the clinical management of spinal cord astrocytomas.IDH1, that the MGMT promoter methylation rate was low, that the BRAF V600E mutation was associated with a good prognosis, and that the TERT promoter mutation was associated with a poor prognosis. Importantly, although the H3 K27M mutation is recognized as a robust marker for diffuse midline gliomas, we demonstrated that the histological grade cannot be ignored when assessing the prognosis of H3 K27M-mutant spinal cord astrocytomas. Altogether, our findings provide evidence-based information for the improved management of spinal cord astrocytomas.We have identified the molecular features of spinal cord astrocytomas in a relatively large cohort of this extremely rare tumor, revealing that spinal cord astrocytomas were mainly wildtype for Additional file 1 : Supplementary Table 1. Primers used in this study