In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations  for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein’s structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed. To reduce vaccine cost and expand immunization coverage, multi-dose formulations are an important component of global immunization programs.,Multi-dose formulations of inactivated or recombinant-protein based vaccines contain antimicrobial preservatives (APs) to prevent the growth of microorganisms that may be introduced accidently during multiple drawings from a single container. The most commonly used vaccine preservative is thimerosal, followed by phenoxyethanol and phenol.et al. observed loss of potency of inactivated poliovirus vaccine (IPV) upon its combination with diphtheria-tetanus-pertussis vaccine containing thimerosal,,,Despite the need for APs to protect multi-dose formulations against microbial contamination, several studies have reported detrimental effects and incompatibility of APs with certain vaccines. For example, Sawyer ,+ T-cell epitope, and are simply referred to as the P[4], P[6], and P[8] protein antigens. The NRRV vaccine candidate has been shown to be safe and immunogenic in infants and toddlers in early clinical trials conducted by PATH in South Africa,,Recent work in our laboratories demonstrated the incompatibility of two APs  with three different recombinant non-replicating rotavirus (NRRV) fusion protein antigens as part of the analytical and formulation development of a trivalent NRRV vaccine candidate.Komagataella phaffii  with an emphasis on their stability in the presence and absence of the AP thimerosal (TH) and the aluminum-adjuvant Alhydrogel (AH). AH-adsorbed NRRV P[4] and P[8] antigens were shown be to incompatible with TH during storage, and the TH-induced destabilization could be mitigated by site-directed mutagenesis of the single cysteine residue. Here, we explore in detail the molecular mechanism(s) by which TH interacts with variants of the NRRV P[4] antigen, using material produced in E. coli and K. phaffii, including the effects of TH on the protein antigen’s structural integrity and conformational stability , antibody-binding properties , and local backbone flexibility . We also utilized HX-MS epitope mapping to describe the interactions of an P[4]-antigen specific mAb with the P[4] antigen to better elucidate the mechanisms of the observed thimerosal-induced destabilization effects. These findings are discussed in the context of ongoing and future challenges associated with AP-induced destabilization of recombinant protein antigens when being formulated as new vaccine candidates for use in LMICs.As described in a companion paper,E. coli expressed parent protein P[4], K. phaffii (P. pastoris) expressed parent protein P[4], and K. phaffii (P. pastoris) expressed P[4] variant (C173S), which are hereafter referred as E. coli P[4], Pp P[4] and Pp P[4]-C173S, respectively. The detailed description of the design of these P[4] mutants as well as the procedures for their expression and purification are described elsewhere,,A total of three different NRRV P[4] fusion-protein antigens were used in these studies including The frozen aliquots of purified P[4] antigens were thawed at room temperature and dialyzed against 150 mM NaCl, 10 mM sodium phosphate, pH 7.2  buffer using 3.5 kDa molecular weight cut-off (MWCO) Slide-A-Lyzer mini dialysis devices at 4°C. A total of 3 buffer exchanges were performed including one overnight exchange. NanoDrop™ 2000 spectrophotometer  was used to determine the concentration of dialyzed samples as described earlier.,,Experimental details of various physicochemical and immunochemical methods used in this study with the P[4] protein antigens, including UV-visible spectroscopy, SDS-PAGE, intact protein MS spectrometry, differential scanning calorimetry (DSC), extrinsic fluorescence spectroscopy, and inhibition ELISA are described in detail elsewhereread 6.8) at 20°C. Triplicate samples were labeled at 6 different time points between 20 and 62,500 s . For P[4]-TH interaction studies, TH was added at a final concentration of 0.01% w/v (0.25 mM) in each sample prior to incubating with deuterated labeling buffer. For epitope mapping studies, a P[4]-specific mAb and P[4] were mixed at a 2:1 molar ratio, such that the final concentration of P[4]-specific mAb in the sample was 1.2 mg/mL. At each specified time point, the hydrogen exchange was quenched by 1:1 dilution with quenching buffer  to the labeled P[4] sample at 0°C. The quenched sample was injected into a liquid chromatography (LC) system for online digestion using an in-house immobilized pepsin column (2.1 × 50 mm),HX-MS experiments were performed using a LEAP H/DX PAL system  coupled with a quadrupole time-of-flight (QTOF) mass spectrometer  using protocols in line with the well-established HX-MS methodologies. HX-MS workflow for mapping epitopes were carried out as described previously with some adaptations.N-terminal prolines. Normalized differential HX The HX data were processed using HDExaminer software . For TH interaction (or epitope mapping) studies, HX data for each peptide from all HX times were averaged into a single differential value representing the TH-(or P[4] specific mAb)-bound P[4] minus P[4] alone. These values were then normalized to their theoretical maximal uptake, which is the length of peptide minus two then minus all non-E. coli P[4] , consistent with the formation of protein-ethylmercury adduct via the only free cysteine, C173, of P[4] protein  value by 9°C as measured by each method  resulted in a substantial decrease in the relative ion abundance of the P[4]-ethylmercury adduct, suggesting reversibility of P[4] and TH interaction  showed a broader, likely multicomponent thermal transition compared to the sharper, likely single component transition observed with the control samples  and E. coli P[4]+TH (black trace)). The heterogeneity observed in E. coli P[4] + TH + dialysis sample could involve both irreversible structural alterations as well as the presence of native and ethylmercury bound protein  is shown in Together, these results point toward the “partially reversible” nature of the TH interaction with P[4]. A working mechanism of P[4]-TH interaction adapted from Trümpler E. coli P[4] and the structural impact of the interaction of TH with E. coli P[4]. A total of 67 peptic peptides were generated and confirmed for E. coli P[4] with 100% sequence coverageE. coli P[4] was then established by analyzing the HX-MS data across six different labeling time points, and the peptides were rank-ordered into three groups based on the hydrogen exchange after 2500 s of labeling time: fast (ΔHX >80%), moderate (20%≤ ΔHX ≤80%), and slow (ΔHX <20%) exchange regions , E52-I64 , F98-M100 (ΔHX rank 6), V122-F134  and I154-F162  showed slowest hydrogen exchange  to better understand the inherent flexibility of  regions . These r regions . As expeexchange  and wereexchange . The resexchange .Fig 3HX-E. coli P[4] with versus without 0.01% TH was performed. No peptides with ethylmercury bound species were observed during the HX-MS analysis of P[4]-TH interaction, likely due to their lower abundance or interference by quench buffer containing 4M guanidinium chloride. The HX differences between E. coli P[4] with versus without the addition of TH at each of the six labeling times between 20 and 62,500 s are shown in N-terminal to the most C-terminal peptides. The HX difference (ΔD) of each peptide was plotted by using the hydrogen exchange in the presence of 0.01% TH minus hydrogen exchange of E. coli P[4] alone. Although no significant differences in uptake were observed at 20 s of labeling time, by 500 s the helical region surrounding C173  showed significant increase in the HX exchange, indicating increased local flexibility in the helical region induced by TH addition. By 2500 s, additional regions covering the β-sheet core, residues V122-S155  also showed significant increase in the exchange. HX of these peptides from these two regions of P[4] remained faster in the presence of TH (compared to the no TH sample) through later labeling times, demonstrating increased flexibility of only these peptides induced by TH ; weak effects (light orange); insignificant effects (gray). It can be seen that the peptide covering residues N174-L181 (peptide #67) was strongly affected due to the interaction between E. coli P[4] and TH, while multiple adjacent peptides covering the surrounding helical region  were weakly affected (100-L121 (peptides #47-52) and V122-S155 (peptides #54-59).The exchange differences for each P[4] peptide at individual time points, in the presence versus absence of TH, were then averaged  validating the C173 residue as the site of TH binding, (2) further understanding the mechanisms of TH-induced destabilization of P[4], and (3) evaluating the extent that the Pp P[4]-C173S is able to resist TH-induced destabilization. The C173S variant was produced in K. phaffii as a part of evaluating the intensified manufacturing platform for low-cost production of recombinant vaccine antigens as described elsewhere.,K. phaffii, and both P[4] proteins (Pp P[4] and Pp P[4]-C173S) were analyzed for changes in the primary structure, higher-order structure, conformational stability, and antibody binding upon addition of TH  showed similar overall physicochemical and antibody-binding properties as well as sensitivity to the addition of the preservative TH . The Pp P[4]-C173S contained full-length species, 20,501 Da, along with several N-terminal truncated species, 20,370 Da, 19,837 Da, and 18,906 Da with average measured masses within 0.3-0.4 Da of the theoretical masses (Pp P[4] (both produced in K. phaffii). In contrast to the parent protein, intact protein mass analysis of Pp P[4]-C173S showed no adduct formation upon addition of TH  to be the same in the presence and absence of TH  and consequently, a slightly lower binding affinity (~3 nM vs. 1 nM) for Pp P[4]-C173S relative to E. coli P[4] and Pp P[4], with no notable changes in binding constants observed upon the addition of TH  were overall similar with a total of 72 and 82 peptic peptides, respectively and with 100% sequence coverage (E. coli P[4] (see above). The inherent flexibility of these two P[4] molecules was established by analyzing the HX-MS data of Pp P[4] and Pp P[4]-C173S in same way as described above for the E. coli P[4]  located in the N-terminal P2 epitope and linker region (highlighted in yellow). Sections of the β-sheet core of Pp P[4], including residues Y38-L41 (peptide #20), V49-I64 (peptides #26-31), F98-M100 (peptide #41), V122-T143 , and I154-F162 (peptide #63), were assigned as the slowly exchanging regions (highlighted in blue)  as relatively fast exchange (highlighted in yellow) while residues Y38-L41 (peptide #22), F59-I64 (peptides #31-34), F98-F101 (peptides #48-49), V122-F134 , and I154-F162 (peptide #73) covering the central β-sheet core of Pp P[4]-C173S were shown to be relatively slowly exchanging (highlighted in blue) (Consistent with in blue) . Similarin blue) .E. coli P[4] and Pp P[4], the Pp P[4]-C173S protein also showed another fast exchange region covering residues I164-E175 (peptide #81) situated in the helical region (E. coli P[4] and Pp P[4] contained low levels of dimer and other HMW species (≤5%) that consisted of non-native intermolecular disulfide bonds between C173 residues of individual monomers. Although such disulfide bonds between two monomers could potentially also result in increased protection (slower HX) near the disulfide region for E. coli P[4] and Pp P[4] (compared to Pp P[4]-C173S), no peptides containing disulfide cross-linked C173 were identified in the HX-MS analysis, possibly due to their overall low abundance after the pepsin digestion under the quenching condition. To further investigate the effect of C173S mutation of P[4], we also determined the HX uptake difference for each shared peptide between Pp P[4] and Pp P[4]-C173S  as the region with significantly greater HX uptake by Pp P[4]-C173S at multiple labeling time points , next to C173, as strongly affected by TH (highlighted in dark orange), while residues Y163-C173 (peptides #68-70), and V122-S155  were affected weakly by TH (highlighted in light orange) . At 12,500 s, however, faster exchange by Pp P[4] + TH was observed in the β-sheet core region , and a larger global increase in hydrogen exchange  was seen by 62,500 s  . Thus, Tcoli see . In contthout TH , a resul62,500 s . In summPp P[4]-C173S showed faster dissociation, and hence relatively weaker binding affinity for P[4]-specific mAb, when compared to E. coli P[4] or Pp P[4] as measured by antibody-antigen binding studies using Octet BLI. Complementary inhibition ELISA studies also displayed a clear shift in the OD450 curve, further confirming weaker antibody binding by Pp P[4]-C173S compared to Pp P[4]  and Y163-L181  of P[4] protein were strongly affected  in the presence of P[4]-specific mAb , F59-I64 (peptide #25), F101-L121 (peptide #42), and I156-F162 (peptides #61-63) were more weakly protected , and Y163-L181 (peptides #68-72) showed strongest protection , F98-G123 , and I157-F162 (peptide #66) displayed weaker protection  and F162-L181  were strongly affected , F98-G123 , and I157-F162 (peptide #76) were weakly affected by P[4]-specific mAb  subunit vaccine candidate, and demonstrated that the three recombinant protein antigens  were destabilized during storage in the presence of the commonly-used vaccine preservative thimerosal.K. phaffii,First, as presented in the companion paper in this issue,E. coli and K. phaffii and C173S variant generated in the latter). Moreover, a combination of antibody-binding studies (Octet BLI) and changes in local backbone flexibility (HX-MS) studies were performed to determine site/regional locations within the P[4] where these effects occur.The second approach, which is the focus of this work, was to elucidate the molecular mechanism(s) of thimerosal interaction with one of the NRRV antigens P[4], including the site of TH-interaction and subsequent effects on the protein’s structural integrity and conformational stability. We used physicochemical methods to examine TH-induced effects on the primary and higher-order structure of the three P[4] antigens  were replaced with carbamidomethylation (Δ57 Da). Second, most of the P[4]-ethylmercury adduct could be removed upon dialysis with PBS buffer, although low level amounts of these adduct species (~5% relative ion abundance) remained even after 6-8 rounds of buffer exchange as detected by intact protein mass analysis. Together, these results indicate reversibility to the formation of P[4]-ethylmercury adduct by addition of competing compounds (IAA) or by concentration gradients .A working mechanism for the P[4]-TH interaction is proposed  based onE. coli P[4] and Pp P[4] as observed by a 9°C reduction in Tm values as measured by DSC and extrinsic fluorescence spectroscopy. After dialysis of the TH-treated P[4] protein, the comparative difference in Tm values (vs. untreated P[4] control) was reduced to 3°C. Together, these results indicate reversibility to the formation of P[4]-ethylmercury adduct, with a likelihood that the TH-induced structural alterations within P[4] protein may be at least partially irreversible in nature.TH-induced modification of Cys173 residue of P[4]  resulted in structural destabilization of E. coli P[4] and Pp P[4], upon addition of TH, an increase in hydrogen exchange around the helical region was observed indicating an increased backbone flexibility of the helix. The middle of this helix is the location of the free cysteine, C173, involved in P[4]-ethylmercury adduct formation. Interestingly, and perhaps more surprisingly, regions of the β-sheet core located far-removed from the ethylmercury coordinated cysteine residue also showed increased hydrogen exchange upon TH addition at longer labeling times. This observation suggests that the structural changes induced by TH were detectable not only at short HX labeling times around the helix region, but also at longer labeling times since the β-sheet core is intrinsically more rigid and thus takes more time to show significant deuterium uptake differences.We used HX-MS analysis to examine the structural elements of the P[4] antigen that are perturbed upon addition of TH. For both in vivo immunogenicity remains to be determined in animal models as part of future work, especially since the nature of the neutralizing epitopes remains unknown . Regardless, a better understanding of preservative-induced destabilization of protein antigens is important to ensure both manufacturing consistency and long-term storage stability of multi-dose recombinant subunit vaccine formulations.These studies demonstrate that TH-induced structural destabilization of P[4] involves increased flexibility in the helical region (containing the ethylmercury modified cysteine residue) as well as more global structural alterations within the P[4] antigen. This global structural destabilization is consistent with the lower conformational stability of the P[4] antigen observed in the presence of TH as measured by DSC and extrinsic fluorescence spectroscopy. Such global structural alterations of the P[4] protein may expose new, otherwise buried, regions of the protein to deleterious interactions that may cause irreversible structural changes in the protein antigen . In fact, such TH-induced interactions are likely the cause for the instability of the Alhydrogel-adsorbed NRRV P[4] antigen in the presence of TH as described in the companion paper in this issue.,,51Cysteine residues are known to play an important role in the structure, biological activity, and conformational stability of proteins.Pp P[4]-C173S) displayed lower conformational stability  compared to E. coli P[4] or Pp P[4]. Concomitantly, the Pp P[4]-C173S protein displayed improved properties in the presence of TH. Upon TH addition, Pp P[4]-C173S did not form ethylmercury adducts, remained resistant to conformational destabilization, and showed no change in local flexibility. Interestingly, the increased flexibility in the helix region of P[4] induced by the cysteine alteration was comparable to the effect of TH modification of the free cysteine residue in E. coli P[4] or Pp P[4]. However, the P[4] parent protein experienced increased levels of structural perturbation, as revealed by increased HX at longer labeling times, indicating increased flexibility throughout the core of protein due to interaction with TH, an effect lacking in Pp P[4]-C173S. This observation likely explains why the P[4] parent proteins in presence of TH  have lower conformational stability than that observed with Pp P[4]-C173S protein .In this work, similar to the observations in literature, an engineered P[4] variant , an additional fast exchange region localized in the helix containing the C173S change was observed with Pp P[4]-C173S. It should be pointed out that when making such comparisons between homologous backbone segments of two protein variants with different amino acid sequences, both backbone dynamics and intrinsic exchange rates should be considered.,168-A177  using a chemical hydrogen exchange rate calculator.,Pp P[4]-C173S had a slower chemical exchange rate (11.0 s-1) than C173 of Pp P[4] (19.6 s-1), and (2) the adjacent N174 residue also had a slower chemical exchange rate for Pp P[4]-C173S versus Pp P[4]. These calculations  in the helix with Pp P[4]-C173S, we can conclude that the observation of faster HX can be attributed to an increase in local backbone flexibility of the helical region due to the cysteine to serine alteration.The decreased conformational stability of shown in  indicatePp P[4]-C173S protein is essentially intermediate between the P[4] parent protein in the absence of TH versus the P[4] parent protein in the presence of TH. Such a rank-ordering was observed in the presence versus absence of TH for the storage stability profiles of Alhydrogel-adsorbed P[4] antigens (parent protein vs. C173S mutant), as described in the companion paper in this issue.Based on these combined data, it can be summarized that the relative conformational stability of the ,,N-terminal P2 linker region, the helical region with C173 in Pp P[4] (S173 in Pp P[4]-C173S), and regions of the central β–sheet core. Since protein-protein interactions often occur across large surfaces,HX-MS analysis can also provide peptide-level structural information to identify the epitope(s) for antigen-antibody interactions.Pp P[4]-C173S protein compared to the P[4] parent protein in the presence of TH . At the same time, as compared to the P[4] parent protein, the Pp P[4]-C173S protein showed weaker binding affinity to the P[4]-specific mAb, however, the TH-modified P[4] parent protein did not display a reduction in P[4]-specific mAb binding affinity. It is possible that this apparent indifference of P[4]-specific mAb binding to P[4] parent protein in the presence of TH may be due to the reversibility observed in TH binding, while changes in the conformation induced by the cysteine to serine mutation may permanently alter certain interactions of P[4] with P[4]-specific mAb, thus, resulting in a relatively weaker binding affinity.We observed the crucial role of the helix encompassing C173 in the binding of P[4]-specific mAb by HX-MS epitope mapping, along with the relatively lower binding affinity of mAb for P[4] upon mutation of C173 by Octet and ELISA. Together, these results support the notion that the cysteine residue of the P[4] plays an important role in stabilizing the P[4] and P[4]-specific mAb complex. Interestingly, similar backbone dynamics was observed for the Pp P[4]-C173S was not affected by the addition of TH, yet showed somewhat lower inherent conformational stability  and several-fold lower antibody binding compared to the P[4] parent protein. Concomitantly, Alhydrogel-adsorbed Pp P[4]-C173S showed improved storage-stability (compared to the parent protein) in the presence of TH as shown in the companion paper.in vivo immunogenicity will ultimately need to be determined with Alhydrogel-adsorbed antigen. Together, such future studies will further our understanding of how different types of preservatives will affect the ability to develop a multi-dose, preservative compatible, aluminum-adjuvant adsorbed NRRV vaccine candidate.The mechanism of thimerosal (TH) interaction with, and TH-induced destabilization of, the NRRV P[4] recombinant protein antigen was investigated through a combination of physicochemical techniques, antibody-antigen binding studies, and local flexibility changes via HX-MS analysis. The interaction between P[4] and TH occurred via the ethylmercury (an aqueous medium degradant of TH) through the C173 residue of P[4]. It occurs essentially instantaneously via a proposed S-Hg coordinate bond, resulting in enhanced local flexibility in the helical regions of P[4] proximal to the ethylmercury coordinated cysteine, followed by a more global structural alteration over time. Although the formation of ethylmercury adduct to the P[4] protein was partially reversible, its destabilizing impact to the structural integrity and conformational stability of P[4] was partially irreversible. Epitope mapping identified the helical region near C173 as a key binding site for P[4] and P[4]-specific mAb interaction, and thus this antibody-antigen binding analysis can be affected by TH addition or cysteine mutation. The


Neuraminidase inhibitors (NAIs) are antiviral agents recommended worldwide to treat or prevent influenza virus infections in humans. Past influenza virus pandemics seeded by zoonotic infection by avian influenza viruses (AIV) as well as the increasing number of human infections with AIV have shown the importance of having information about resistance to NAIs by avian NAs that could cross the species barrier. In this study we introduced four NAI resistance-associated mutations (N2 numbering) previously found in human infections into the NA of three current AIV subtypes of the H5Nx genotype that threaten the poultry industry and human health: highly pathogenic H5N8, H5N6 and H5N2. Using the established MUNANA assay we showed that a R292K substitution in H5N6 and H5N2 viruses significantly reduced susceptibility to three licenced NAIs: oseltamivir, zanamivir and peramivir. In contrast the mutations E119V, H274Y and N294S had more variable effects with NAI susceptibility being drug- and strain-specific. We measured the replicative fitness of NAI resistant H5N6 viruses and found that they replicated to comparable or significantly higher titres in primary chicken cells and in embryonated hens’ eggs as compared to wild type - despite the NA activity of the viral neuraminidase proteins being reduced. The R292K and N294S drug resistant H5N6 viruses had single amino acid substitutions in their haemagglutinin (HA): Y98F and A189T, respectively (H3 numbering) which reduced receptor binding properties possibly balancing the reduced NA activity seen. Our results demonstrate that the H5Nx viruses can support drug resistance mutations that confer reduced susceptibility to licenced NAIs and that these H5N6 viruses did not show diminished replicative fitness in avian cell cultures. Our results support the requirement for on-going surveillance of these strains in bird populations to include motifs associated with human drug resistance. •A R292K residue change in NA reduces susceptibility to NA inhibitor drugs (NAIs) in H5N6 and H5N2 avian influenza viruses.•All four mutations  reducing susceptibility to NAIs in H5N6 viruses reduced viral NA activity.•Reduced susceptibility to NA inhibitors in H5N6 did not attenuate virus replication efficiency in chicken cells or eggs.•A reduction of the viral HA affinity for sialic acid was observed in H5N6 viruses with reduced NA activity. There are multiple genotypes of AIV categorised on the possession of one of 16 known surface haemagglutinin (HA) proteins and 9 neuraminidase (NA) proteins. AIVs frequently transmit from these reservoir species to other bird populations such as domestic poultry, from where they can cross additional species barriers and jump into mammalian hosts such as humans. An increase of human infections caused by different AIV subtypes in recent years , 2019 toIn the absence of universal influenza virus vaccines, antivirals are important treatment and prophylaxis tools in the mitigation of AIV in humans. Whilst there has been recent progress in the development of novel influenza anti-viral drugs, such as polymerase inhibitors - represented by baloxavir marboxil approved in 2018 , the curAntiviral drug resistance of influenza virus is known to develop following treatment of humans with the NAI drugs for both human influenza strains (H1N1 and H3N2) and AIVs (H7N9 and H5N1) . Indeed,The error prone polymerase of the influenza virus results in natural diversity that may, albeit rarely, in the absence of a drug selection pressure result in natural NAI resistance by AIVs. An analysis performed by Understanding the signatures that confer resistance to antiviral drugs for currently circulating strains of virus is therefore important information. Determination of whether resistance motifs alter the fitness of the viruses that possess them is also of importance - it is likely that viruses prevalent in migratory birds could be transmitted to domesticated poultry and thus subsequently find their way across the avian – mammalian host barrier. The fitness of influenza A viruses relies in part on the functional balance between the two surface glycoproteins: HA, which binds to cell surface sialic acid to enable attachment and entry of the virus to cells; and NA, which cleaves sialic acid to allow egress and dissemination of the new virions to additional target cells or a new host organism. The 9 NA subtypes found in AIV isolates are phylogenetically split into two groups: Group 1 comprising N1, N4, N5 and N8, and Group 2 including N2, N3, N6, N7 and N9 .In the past five years a lineage of highly pathogenic H5 viruses, known as the H5Nx viruses, have caused multiple worldwide outbreaks in poultry. The H5Nx viruses all possess the same clade 2.3.4.4 H5 HA protein of the A/goose/Guangdong/1/1996 highly pathogenic H5N1 virus origin . ReassorIn this study we introduced four NAI resistance-associated mutations  into three recent H5Nx viruses containing different avian NA subtypes: N2, N6 and N8. To maintain optimal HA/NA balance during replication in avian substrate all recombinant viruses carried H5 HA with multibasic cleavage site of the original strain. We determined the susceptibility of these viruses to three licensed NAIs: OSE, ZAN and PER. Furthermore, for the H5N6 subtype , we assessed virus replication efficiency of selected drug-resistant strains in primary chicken kidney cells (cKC) and embryonated hen's eggs, and compared the NA activity and HA receptor binding to viruses carrying a wild type (wt) NA.22.1Madin-Darby Canine Kidney  and human embryonic kidney cells  were maintained as described previously . PrimaryGISAID accession nos. EPI661558 and EPI661559); A/scarlet_ibis/Germany/AR44-L01279/2015 (GISAID accession nos. EPI624533 and EPI624535) (H5N8); A/goose/Taiwan/01031/2015 (H5N2) (and KU646885GenBank accession nos. KU646887 . Single nucleotide changes in NA were introduced using QuikChange™ site-directed mutagenesis protocol (Stratagene). For NA activity assay H5N6 NAs with C-terminal FLAG tag were subcloned into pCDNA3.1(+) vector (ThermoFisher). All recombinant viruses were generated using an eight plasmid reverse genetics system (The following NA and HA genes were synthesised by GeneArt (Invitrogen) and cloned into a pHW2000 vector :A/chickes system  with 6 is system . Virusess system . The HA s system  with chis system . The intAll viruses carrying H5 HA with multibasic cleavage site and/or NA modified to increase the resistance to licensed NAIs were handled in the SAPO4/BSL3 containment facility of The Pirbright Institute by trained and authorised personnel.2.22, 1% FCS; pH 6.5) for live-cell NA activity assay. Protein lysates were analysed by Western blot with α-FLAG (Sigma-Aldrich) and α-GAPDH (Invitrogen) antibodies, and the band intensities were quantified using ImageJ software : normal inhibition (NI) < 10-fold; reduced inhibition (RI) 10 to 100-fold; highly reduced inhibition (HRI) > 100-fold. , zanamivir  and peramivir  as described elsewhere . Virus d00-fold. .2.4Infectious virus from harvested allantoic fluid or cell supernatant was titrated by plaque assay on MDCK cells as described before , with mo2.550 represents half maximal effective concentration of drug leading to 50% reduction in plaque size as compared to no drug. The data was analysed in Excel (MS Office) and the EC50 values were calculated using Graph Pad Prism software .Plaque reduction assay (PRA) on MDCK cells was performed as described elsewhere , with mo2.6in vitro, cKC cells in 6-well plates were inoculated with virus at an MOI of 0.001 (4000 PFU/ml), each virus in triplicate as described previously  of virus in PBS supplemented with 1% penicillin/streptomycin and 0.35% BSA as described previously . 4 HAU of each virus was added to sialidase-treated 1% [v/v] cRBCs solution and incubated for 1 h at 4 °C. The highest enzyme concentration allowing for full virus haemagglutination was recorded [U/ml] and expressed as a relative receptor binding avidity.Receptor binding avidity assay of recombinant viruses was performed as described elsewhere , with mo2.8Viral RNA (vRNA) was extracted and purified from allantoic fluid using QIAmp Viral RNA kit (Qiagen) and cDNA was synthesised using Verso cDNA reverse transcription kit (ThermoFisher Scientific) and influenza universal primers . The HA 2.9MDCK and cKC were cultured for 72 h in the presence or absence of NAI drugs, each condition in triplicate. Relative cell viability was quantified based on the presence of ATP as an indicator of metabolically active cells using CellTiter-Glo® Luminescent Assay (Promega) according to manufacturer's protocol.2.10www.minitab.com) using general linear model with post hoc Dunnett's test  or Kruskal-Wallis and Mann-Whitney nonparametric tests (growth curve in eggs).Statistical analyses were performed in Minitab® Statistical Software  are E119V, H274Y, R292K and N294S (N2 NA subtype numbering) . Two of 3.250 values. IC50 is the concentration of drug that will reduce the NA activity by 50% from no drug , zanamivir (ZAN) and peramivir (PER) using the MUNANA fluorescence-based NA inhibition assay to generate IC no drug . With thted host . It was y others .Fig. 2Ne50 values for the E119V mutation in all three viruses demonstrated RI, the H274Y mutation resulted in RI for the H5N6 NA and HRI for the H5N8 NA, and N294S mutation showed RI only in H5N8 background  completely abrogated plaque formation of wt H5N6, whereas no reduction in plaque size was observed for H5N6_R292K  and assessed the replicative capacity of the H5N6 AIV subtype bearing NAI resistance-associated markers. Three H5N6 mutant viruses, E119V, N294S and R292K showed reduced susceptibility to at least one drug , and desWe inoculated primary cKC at an MOI of 0.001 PFU/cell with each virus and collected the supernatants at the indicated time points C. All th4 PFU/ml, which was ~2-log more than wt H5N6 and R292K, however the differences were non-significant (7–1.5 × 108 PFU/ml (4 PFU/ml) and reached the maximum of 1.4 × 107 PFU/ml only at 48 h p.i. The differences in virus titres for all three mutants  were non-significant (p > 0.05) as compared to wt H5N6 with exception of R292K at 24 hpi which replicated to significantly higher titres (p < 0.01).To verify the effects we observed in cKCs we inoculated 10-day old embryonated hen's eggs with drug-resistant H5N6 viruses, sampled the eggs at indicated time points and determined the median titres of infectious virus  D – F. Anificant D. At 24 8 PFU/ml E, and th8 PFU/ml F, when ain vitro and in ovo fitness assessment we concluded that the NAI-resistant H5N6 viruses are able to replicate at least as efficiently as the wild type in tested models.Pre-incubation of wt H5N6 with 100 μM PER significantly reduced viral titres at 48 h p.i. when compared to eggs inoculated with virus only (p < 0.05), but it was not enough to completely abrogate virus production D–F. The 3.5Concurrent mutations in HA can re-balance changes affecting NA activity therefore we analysed the HA sequences of NAI-resistant H5N6 viruses propagated in eggs. Two H5N6 mutants acquired single amino acid substitutions in their HA following rescue and stock preparation in eggs: Y98F (H3 numbering) in the R292K NA mutant and A189T in N294S NA mutant virus A. Both m4In this study we investigated whether known resistance mutations that occur in human isolated IAV strains  function to reduce the susceptibility to NAI drugs in the highly pathogenic H5Nx viruses  that have been responsible for large scale poultry outbreaks in recent years. In contrast to previous reports investigating functional NAI resistance signatures in AIV carrying 7 genes (including HA) from laboratory strain PR8 (H1N1)  we used The four mutations: E119V, H274Y, R292K and N294S did therefore confer variable reduced drug susceptibility to the NA proteins of highly pathogenic H5N2, H5N6 and H5N8. Some mutations such as E119D/Q, H274Y/R or N294S, albeit rare in avian isolated H5Nx viruses, were found to have naturally arisen  and we wUsage of the oral drug OSE has increased dramatically for the treatment and prophylaxis of seasonal human influenza infection in Japan and the USA since the swine flu pandemic of 2009 . The actWe also know from previous experience that unregulated use of human anti-viral drugs in farmed animals can occur by poultry farmers under pressure from a constant avian influenza threat. In the late 1990s there is evidence of the unregulated use of the anti-viral compound amantadine in farmed poultry in China . Subsequin vivo where mucus provides a barrier to infection and transmission (Known mutations that confer reduced susceptibility to the NAI drugs are in the active site of NA and the associated framework residues that maintain the active site structure. We and others have shown that the NA mutations which result in reduced susceptibility to the NAI drugs often also have a reduced NA activity compared to the wt NA viruses and this can impact viral replication fitness, particularly smission . Therefosmission . Our ressmission . Prior tsmission . It has smission . We obsesmission . Concurrsmission . This isIn summary, our study has shown that the NAI resistance-associated mutations commonly found in human influenza A strains also reduce the susceptibility to those drugs in recently emerged avian H5Nx viruses. These mutations can reduce viral NA activity and prompt the virus to acquire additional changes to allow for efficient replication in host cells, e.g. by changing the receptor binding avidity of viral HA. Changes in HA can directly affect virus host tropism or facilitate immune escape which may impact zoonotic potential of the H5Nx AIV viruses.10.13039/501100000268BBSRC grant Institute Strategic Programme Grants BBS/E/I/00007037 and BBS/E/I/00007039. The funders had no role in study design, data collection, data interpretation, or the decision to submit the work for publication.This work described herein was funded by The work was conceptualised by HS and DB. Experimental work was executed by DB. The manuscript was written and edited by DB and HS.None declared.


Objective: The microsatellite status and tumor immune microenvironment have a remarkable influence on tumor immunotherapy. This study was performed to investigate programmed cell death protein 1/programmed death ligand 1 (PD1/PDL1) expression and their correlations with CD8+ T cell/CD68+ macrophage (CD68+ M) densities in gastric cancer (GC) at different microsatellite statuses.Methods: The expression of MLH1, PMS2, MSH2, and MSH6 was detected via immunohistochemistry (IHC) to determine the microsatellite status in 215 GC samples obtained from surgical resections. Furthermore, the expression of PD1, PDL1, CD8, and CD68 was detected in the samples via IHC, and the differences and correlations in GC at different microsatellite statuses were then analyzed. PDL1 expression in tumor cells was labeled as PDL1[T], while expression of PD1 and PDL1 in tumor-infiltrating immune cells was labeled as PD1 and PDL1, respectively. Kaplan-Meier analysis was used to evaluate the significance of PD1/PDL1 expression in determining overall survival. Multivariate Cox regression analysis was performed using SPSS software. P-values were determined using the log-rank test.Results: Our results indicated that PD1, PDL1[T], and PDL1 positivity rates were 59%, 35%, and 57% in 46 microsatellite unstable (MSI) GCs and 45%, 22%, and 40% in 169 microsatellite stable (MSS) GCs, respectively. Compared with MSS GC, PD1, PDL1[T], and PDL1 expression was higher in MSI GC . Additionally, CD8+ T cell and CD68+ M densities were higher in MSI GC than in MSS GC . Additionally, CD8+ T cell/CD68+ M densities were evaluated according to tumor center and invasion front. We found that PD1 expression was significantly correlated with CD8+ T cell density at the invasion front of the MSI GC (P = 0.031), whereas PDL1 expression was significantly correlated to high CD68+ M density in the tumor center and invasion front of MSS GC . Survival analysis showed that patients with PD1-positive and PDL1[T]/PDL1-negative GC had better prognosis . Multivariate Cox survival analysis showed that PDL1[T] was an independent prognostic factor for GC.Conclusion: The results suggested that PD1/PDL1 expression and immune response varied at different microsatellite statuses in GC. PD1/PDL1 expression was correlated with CD8+ T cell/CD68+ M densities in GC at different microsatellite statuses, especially at the invasion front. The patients exhibiting high PD1/PDL1 expression or high CD8+ T cell/CD68+ M densities MSI GC might be potential beneficiaries of PD1/PDL1 immunotherapy. Gastric cancer (GC) is the fifth most frequently diagnosed malignancy and the third leading cause of cancer-related death worldwide Mounting evidence has shown that the tumor microenvironment plays an essential role in the progression of many solid tumors; tumor microenvironment is mainly orchestrated by tumor-infiltrating immune cells, including neutrophils, dendritic cells, macrophages, eosinophils, mast cells, and lymphocytes. The tumor microenvironment is indispensable for tumor progression, fostering proliferation, survival, and migration As one of the most promising immunotherapy approaches, programmed cell death protein 1 (PD1) and its ligand, programmed death ligand 1 (PDL1), have been used in the treatment of a variety of tumors in recent years So far, the relationship among MSI characteristics of tumors, heterogeneity of the tumor microenvironment, and PD1/PDL1 immunotherapy has attracted increasing attention in clinical studies. A basic consensus has been reached to identify MSI status and PD1/PDL1 expression to select patients who would benefit from PD1/PDL1 immunotherapy The samples and data of 215 patients who underwent surgical resection of primary GC at the first hospital of China Medical University and Jinzhou Medical University of Liaoning province between January 2012 and December 2018 were collected retrospectively. None of the patients had undergone neoadjuvant therapy. The histological diagnosis was confirmed by two pathologists independently, according to the World Health Organization criteria. Pathological Pathological Tumor Node Metastasis (TNM) staging was classified based on the Union for International Cancer Control . The study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University and Jinzhou Medical University. All participants enrolled in the study signed written informed consent forms.The total number of cases in this study was 215. For each case, 9 consecutive sections were made for HE (Hematoxylin - Eosin) staining and immunohistochemical staining of 8 indexes including MLH1, MSH2, PMS2, MSH6, CD8, CD68, PDL1 and PD1. Because a few cases tissue is too little to the last 2-3 tissue section to complete the immunohistochemical evaluation. So some individual index staining is less than the total number of all cases. MLH1, MSH2, MSH6 and PMS2 evaluations were completed in all 215 cases, and 205 cases were evaluable for PD1, 202 cases for PDL1 [T]/PDL1, 205 cases for CD8, and 202 cases for CD68.The primary antibodies used in this study , DAB chromogenic solution, and UltraSensitive TMSP detection kit were purchased from Maixin . Anti-PDL1 antibody IHC was performed on 4 μm-thick tissue sections mainly as previously described Each section was first observed under a low-power microscope for the entire area. Positive staining areas were selected and observed under a high-power microscope. The evaluation was independently conducted by two senior pathologists.MLH1, PMS2, MSH2, and MSH6 were all located in the GC cell nucleus. The tumor cell nuclei in brown yellow or brown granules were determined to be positive staining. Absence was determined when the tumor cell nucleus failed to stain. Microsatellite stability was determined when four proteins were positive, whereas MSI was determined when one or more proteins were absent. The microsatellite status was determined consistently by evaluating the expression of MLH1, PMS2, MSH2, and MSH6 in GC and adjacent peritumoral tissues simultaneously, although there were significant differences in the expression levels between them.2) from each sample, and manually counted 100 tumor or immune cells in each HPF and then calculated the percentage of positively stained cells. The mean value was adopted as the positive expression rate of PD1, PDL1[T], and PDL1. Positive staining was defined as more than 1% stained cells, regardless of cytoplasmic or membrane staining PD1 and PDL1 were located in the membrane and/or cytoplasm. PDL1 expression in tumor cells was labeled as PDL1[T], and expression of PD1 and PDL1 in tumor-infiltrating immune cells was labeled as PD1 and PDL1, respectively. We randomly selected five high-power fields  in the tumor center and invasive front, respectively, for each case, then manually counted and calculated the average number of stained CD8+ T cells and CD68+ M according to the median number of stained cells . Patients were divided into two groups: high and low density.We randomly selected five HPFs . Intergroup comparison  was conducted using the χ2 test or Fisher's exact test. Kaplan-Meier analysis was used to evaluate the significance of different factors in determining overall survival. Multivariate Cox regression analysis was performed using SPSS software. P-values were determined using the log-rank test. P <0.05 was considered statistically significant.Based on the expression of MLH1, PMS2, MSH2, and MSH6, 215 samples were divided into two groups: 46 MSI (21.4%) and 169 microsatellite stable (MSS) (78.6%) , while the PD1/PDL1[T] expression rate in MSI GC was higher than in MSS GC but without statistical significance (P = 0.109 and 0.090) , patients were divided into two groups: high and low density. CD8+ T cell density in MSI GC was higher than in MSS GC but without statistical significance (P = 0.537). CD68+ M density in MSI GC was significantly higher than that in MSS GC (P < 0.001) . PDL1 expression rate in the high CD68+ M density group was greater than that in the low-density group in MSS GC P = 0.013). However, PDL1[T] expression was not correlated with CD8+ T cell/CD68+ M densities in MSI or MSS GC (Table . HoweverPD1 expression was significantly correlated with CD8+ T cell density at the invasion front of MSI GC P = 0.031). PDL1 expression was significantly correlated with high CD68+ M density in the tumor center and invasion front of MSS GC . However, PDL1[T] expression was not correlated with CD8+ T cell/CD68+ M densities in the tumor center and invasion front of MSI or MSS GC . There were no correlations between CD8+ T cell/CD68+ M densities and prognosis in GC . Multivariate survival analysis showed that microsatellite status, CD8+ T cell density, PDL1[T], TNM stage, infiltration depth, and vascular thrombi were all independent prognostic factors for GC.In this study, we investigated PD1/PDL1 expression and its correlations with infiltrating immune cells in GC with different microsatellite statuses. It was found that both PD1/PDL1 expression and CD8+ T cell/CD68+ M densities were higher in MSI GC than in MSS GC. Furthermore, PD1/PDL1 expression was significantly correlated with high CD8+ T cell/CD68+ M densities, especially at the invasion front. These results may provide new information regarding the accurate stratification of target populations for PD1/PDL1 immunotherapy in GC.A microsatellite is a simple, repetitive sequence evenly distributed in eukaryotic genomes under normal conditions. MSI refers to any change in the length of microsatellites resulting from insertion or deletion of repetitive units in a tumor and usually arises from germline mutations in the components of MMR genes  The tumor microenvironment is primarily composed of TILs, especially CD8+ T cells and CD68+ TAMs. It has previously been reported that tumors with different microsatellite statuses may exhibit different immune microenvironments. MSI is associated with higher density TILs, possibly in response to the new environment created by neoantigens In our study, we analyzed the correlations between PD1/PDL1 expression and CD8+ T cell/CD68+ M densities in MSI or MSS GC. We found that PD1 expression was correlated with CD8+ T cell density in MSI GC, and PDL1 expression was significantly correlated with high CD68+ M density in MSS GC. It has been reported that the most common tumor-infiltrating immune cells are CD8+ T cells and CD68+ M, accounting for 15% and 13% of all tumor cells, respectively. Among the four molecular subtypes of GC classified by TCGA, MSI GC shows high-density infiltration of T cells and macrophages, among which T cells account for 30%-50%, while macrophages account for 20%, and 70% of MSI tumors express PDL1 The heterogeneity of immune cell distribution within tumors can influence the therapeutic response This study further analyzed the relationship between PD1/PDL1 expression and CD8+ T cell/CD68+ M densities and prognosis. Until now, PD1/PDL1 expression in relation to prognosis in GC remains elusive. A meta-analysis showed that PDL1 negativity was associated with better overall survival in GC In summary, we evaluated PD1/PDL1 expression and its correlation with CD8+ T cell/CD68+ M densities in MSI or MSS GC. The results showed that PD1/PDL1 expression and CD8+ T cell/CD68+ M densities were higher in MSI GC than in MSS GC, suggesting that PD1/PDL1 expression and the immune response varied based on microsatellite status in GC. Moreover, we found a significant correlation between PD1/PDL1 expression and high CD8+ T cell/CD68+ M density, especially at the invasion front in MSI or MSS GC, suggesting that they may participate in immune escape and affect the efficacy of immunotherapy. Patients with MSI with high PD1/PDL1 expression or high CD8+ T cell/CD68+ M densities might be potential beneficiaries of PD1/PDL1 immunotherapy. This study may provide a better understanding of the immune microenvironment in GC with different microsatellite statuses and may provide clues for seeking potential therapeutic targets and prognostic biomarkers for the treatment of GC in clinical settings.


Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing. Trypanosoma brucei is essential for variant surface glycoprotein (VSG) monoallelic expression, an important aspect of antigenic variation, where T. brucei regularly switches its major surface antigen, VSG, to evade the host immune response. Like other RAP1 orthologs, T. brucei RAP1 (TbRAP1) has conserved functional domains, including BRCA1 C terminus (BRCT), Myb, MybLike, and RAP1 C terminus (RCT). To study functions of various TbRAP1 domains, we established a strain in which one endogenous allele of TbRAP1 is flanked by loxP repeats, enabling its conditional deletion by Cre-mediated recombination. We replaced the other TbRAP1 allele with various mutant alleles lacking individual functional domains and examined their nuclear localization and protein interaction abilities. The N terminus, BRCT, and RCT of TbRAP1 are required for normal protein levels, while the Myb and MybLike domains are essential for normal cell growth. Additionally, the Myb domain of TbRAP1 is required for its interaction with T. brucei TTAGGG repeat-binding factor (TbTRF), while the BRCT domain is required for its self-interaction. Furthermore, the TbRAP1 MybLike domain contains a bipartite nuclear localization signal that is required for its interaction with importin α and its nuclear localization. Interestingly, RAP1’s self-interaction and the interaction between RAP1 and TRF are conserved from kinetoplastids to mammals. However, details of the interaction interfaces have changed throughout evolution.RAP1 is a telomere protein that is well conserved from protozoa to mammals. It plays important roles in chromosome end protection, telomere length control, and gene expression/silencing at both telomeric and nontelomeric loci. Interaction with different partners is an important mechanism by which RAP1 executes its different functions in yeast. The RAP1 ortholog in IMPORTANCETrypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, to evade the host immune response. VSGs are expressed from subtelomeres in a monoallelic fashion. TbRAP1, a telomere protein, is essential for cell viability and VSG monoallelic expression and suppresses VSG switching. Although TbRAP1 has conserved functional domains in common with its orthologs from yeasts to mammals, the domain functions are unknown. RAP1 orthologs have pleiotropic functions, and interaction with different partners is an important means by which RAP1 executes its different roles. We have established a Cre-loxP-mediated conditional knockout system for TbRAP1 and examined the roles of various functional domains in protein expression, nuclear localization, and protein-protein interactions. This system enables further studies of TbRAP1 point mutation phenotypes. We have also determined functional domains of TbRAP1 that are required for several different protein interactions, shedding light on the underlying mechanisms of TbRAP1-mediated VSG silencing. Telomeres are nucleoprotein complexes at linear chromosome ends. They are essential for genome integrity and chromosome stability . TelomerSaccharomyces cerevisiae RAP1 (ScRAP1) achieves different goals through interactions with various protein partners  domain (–RAP1 C terminus (RCT)  domain , 12, 16 ) domain , 61 and ) domain –64. Humaus (RCT) , which i(RCT) 56––68. All ) 56––11–, 16, 69.directly .Trypanosoma brucei , thereby effectively evading the host immune response  was identified as a factor interacting with T. brucei TTAGGG repeat-binding factor (TbTRF) , an increased amount of telomeric RNA:DNA hybrids, and an elevated amount of telomeric/subtelomeric DNA damage  , which b (TbTRF) . TbRAP1 000-fold , 44. Trarequency . AdditioA damage . We showterminus . Here, wTbRAP1 allele is flanked by two loxP repeats so that it can be conditionally deleted by inducing Cre expression. Using this system, we determined that TbRAP1 Myb is necessary for TbTRF interaction and VSG silencing. Using TbRAP1 MybLike as bait in a yeast 2-hybrid screen, we have determined that importin α interacts with TbRAP1’s nuclear localization signal (NLS) residing in the MybLike domain. We found that TbRAP1 interacts with itself through the BRCT domain. In addition, the N terminus, BRCT, and RCT of TbRAP1 are required for normal TbRAP1 protein levels, while Myb and MybLike are essential for normal cell growth. These results not only provide further evidence of conserved essential functions of RAP1 orthologs throughout evolution but also pave the way for a better understanding of the mechanisms explaining how TbRAP1 silences subtelomeric VSG genes and helps maintain telomere stability and integrity.In this study, we established several strains in which one endogenous TbRAP1 is an essential protein  and a loxP site with the blasticidin resistance gene (BSD) were targeted to locations upstream and downstream, respectively, of a given TbRAP1 allele (TK) from the herpes simplex virus, allowing selection for cells that had lost the floxed TbRAP1 allele (denoted as “F”) by the use of ganciclovir (GCV), as expression of the TK gene in T. brucei renders the parasite sensitive to GCV (GFP) with both selectable markers  in two TbRAP1F/+ clones  (see Fi10.1128/mSphere.00027-20.1FIG S1TbRAP1. (A) Western blotting using a rabbit GFP antibody (Life Technologies) to detect HYG-GFP-TK and BSD-GFP-TK expression in TbRAP1F/+ (-Cre-Ep1) cells before and after transient transfection of Cre-EP1 (TbRAP1F/+ (-Cre-Ep1) cells before and after transient transfection of Cre-EP1  Growth curves of two clones of TbRAP1F/+ cells (with Cre-EP1 targeted to an rDNA spacer region) before and after induction of Cre by doxycycline. Cells were selected with different combinations of antibiotics as indicated. The Cre-EP1 plasmid has a phleomycin resistance (BLE) marker  Sou Cre-EP1 . A DNA f) marker . DownloaCopyright © 2020 Afrin et al.2020Afrin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TbRAP1 allele can be excised by Cre efficiently, we transiently transfected the Cre-EP1 plasmid into TbRAP1F/+ (-Cre-EP1) cells. Pools of cells and individual clones were selected with GCV. Western analysis performed with the GFP antibody showed that HYG-GFP-TK and BSD-GFP-TK were no longer expressed in either the pool or the clones  spacer region, whose expression can be induced by doxycycline  , and two sets of TbRAP1 primers were used: one annealed specifically to the N-terminal region and another annealed to the BRCT domain (TbRAP1 mRNA level dropped to 10% of the wild-type (WT) level in TbRAP1F/− cells  . In the  in 48 h , confirmT domain . After 2/− cells , indicat/+ cells , as onlyTbRAP1 by RNAi led to cell growth arrest and VSG derepression  were derepressed by several 10-fold orders after induction of Cre for 24 h and by several 100-fold orders after 48 h -tagged TbRAP1ΔMyb did not express well, we replaced the WT TbRAP1 allele with a TbRAP1F2H-ΔMyb mutant  The rabbit TbRAP1 antibody (6-TbRAP1-MybLike fragment. (Right) Western blotting of the same recombinant fragment using the rabbit TbRAP1 antibody. (C and D) Classification of genes affected in Cre-induced TbRAP1F/F2H-ΔMyb cells. Distributions  of different types of genes whose mRNA levels were upregulated (C) or downregulated (D) are shown. The number of genes affected in each category is listed in parentheses. Download FIG S2, EPS file, 2.7 MB.(A) Southern analysis confirmed the antibody  used in Copyright © 2020 Afrin et al.2020Afrin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00027-20.7TABLE S1T. brucei strains used in the current study. Download Table S1, DOCX file, 0.01 MB.Copyright © 2020 Afrin et al.2020Afrin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ScRAP1 has both transcription activation and repression functions  RNA was purified and used for library construction at Novogene followed by paired-end high-throughput sequencing using Illumina . Differential gene expression analysis showed that more than 8,000 genes were upregulated and nearly 3,000 genes were downregulated in Cre-induced TbRAP1F/F2H-ΔMyb cells compared to the results seen with TbRAP1F/+  expression sites (BESs) (VSG genes and some BES-linked expression site-associated genes (ESAG genes) were upregulated, but other BES-linked ESAG genes were not affected or were even downregulated   showed tegulated . Based oegulated , which i7 genome . Therefoal level . It is p10.1128/mSphere.00027-20.3FIG S3TbRAP1F/+ and TbRAP1F/F2H-ΔMyb cells. RNA samples were isolated from three independently induced cells in both strains. RNA sequence reads were mapped to the T. brucei Lister 427 genome TriTrypDB-45_TbruceiLister427_2018_Genome.fasta, which was obtained from TriTrypDB. Read coverage for each sample was viewed in IGV. Each BES is labeled, and the genes inside each BES are listed at the bottom. The whole BES region is shown with the promoter located on the left side. The y axis data represent the number of RNA reads. The range is indicated on the left of each trace. BES1 was fully active in the cells analyzed. Download FIG S3, PDF file, 1.2 MB.RNA sequence read coverage in all VSG bloodstream-form expression sites (BESs) for Cre-induced Copyright © 2020 Afrin et al.2020Afrin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TbRAP1 allele with an F2H-tagged TbRAP1 mutant lacking the MybLike domain (TbRAP1ΔMybL). Southern blotting confirmed its genotype (TbRAP1ΔMybL was expressed (TbRAP1 (https://myhits.isb-sib.ch/cgi-bin/motif_scan) . F2H-TbRAP1ΔMybL lacks this NLS, which is likely why this mutant is localized in the cytoplasm. To confirm this, we added the simian virus 40 (SV40) large T NLS to the N terminus of TbRAP1ΔMybL  , we founAP1ΔMybL . SoutherWT level C and D.  nucleus .10.1128/mSphere.00027-20.4FIG S4TbRAP1F/F2H-ΔMybL (A) and TbRAP1F/F2H-NLS-ΔMybL (C) cells. Genomic DNA was digested with EcoRI and probed with a DNA fragment amplified by PCR with the forward primer (5′-GCGCCTCGAGACCGACGTTGACGGTG-3′) and the backward primer (5′-GCGCTCTAGAACACGAAGAAATCGGGACGTG-3′) using TbRAP1 as the template. Western analyses showed the expression levels of F2H-TbRAP1ΔMybL (B) and F2H-NLS-TbRAP1ΔMybL (D) in the same clones as those indicated in panels A and C, respectively. Download FIG S4, EPS file, 1.1 MB.Southern analyses confirmed the genotypes of Copyright © 2020 Afrin et al.2020Afrin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TbRAP1 is imported into the nucleus, we screened a normalized yeast 2-hybrid library generated from T. brucei cDNA using the TbRAP1 MybLike domain as bait. In this screen, 16.5 million yeast primary transformants were obtained, and a total of 711 clones were positive in the initial screen. The majority of the candidates represented the same gene, Tb427.06.2640, which is annotated as encoding the importin α subunit in TriTrypDB  epitope at one endogenous importin α allele. PCR analysis confirmed correct targeting in three different TbRAP1 backgrounds: both alleles were WT, one of the two WT alleles had an N-terminal F2H tag, and one of the alleles was replaced with the TbRAP1F2H-ΔMybL mutant  and TbRAP1F2H-ΔMybL/+ TRFi cells, IP experiments were performed using HA monoclonal antibody 12CA5  or 9E10. In cells carrying untagged or F2H-tagged WT TbRAP1, both importin α-myc13 and TbRAP1 were present in the IP products (TbRAP1ΔMybL and importin α-myc13 were not in the same IP product (TbRAP1 (and most likely the NLS in this domain) is necessary for TbRAP1’s interaction with importin α and its nuclear localization.To confirm the interaction between importin α and L mutant . The latblotting . Subsequantibody  or the m10.1128/mSphere.00027-20.5FIG S513 tag. Genomic DNAs from the parent TbRAP1+/+ strain (as a negative control) and three transfected cells in the TbRAP1+/+, TbRAP1F2H+/+ TRFi, and TbRAP1F2H-ΔMybL/+ TRFi background were used as the templates. Forward primer (5′-CACTTTGTCTCCTCGAGACA-3′) and backward primer (5′-CGATCAGAAACTTCTCGACAG-3′) were used in the PCR amplification. A product of 1.26 kb is expected for correct targeting of the myc13 tag. Download FIG S5, EPS file, 1.2 MB.PCR confirmed the endogenous tagging of importin α with a C-terminal mycCopyright © 2020 Afrin et al.2020Afrin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TbRAP1 was originally identified as a TbTRF-interacting factor in a yeast 2-hybrid screen  is sufficient to interact with TbTRF in yeast 2-hybrid analysis  is essential for the TbRAP1-TbTRF interaction, in the TbRAP1F/+ cells, we either targeted an F2H tag to the N terminus of the WT TbRAP1 allele (to generate the TbRAP1F/F2H+ strain) or replaced the WT allele with an F2H-tagged TbRAP1 mutant lacking various functional domains (to generate the TbRAP1F/F2H-mut strains)  at the C terminus of TbRAP1ΔMybLΔRCT. These TbRAP1 mutants were expressed  . Souther strains  to C. Hexpressed  but at m  Southern analyses confirmed the genotype of TbRAP1F/F2H-ΔNT (A), TbRAP1F/F2H-ΔBRCT (B), and TbRAP1F/ΔMybLΔRCT-F2H-NLSr (C) cells. Genomic DNAs were digested with XbaI and BamHI in the experiments represented in panels A and C and with EcoRI in the experiments represented in panel B. A DNA fragment containing the TbRAP1 sequence between the BRCT domain and the Myb domain was used as the probe in the experiments represented in panels A and C. A DNA fragment containing the N-terminal region of TbRAP1 was used as the probe used in the experiments represented in panel B. (D to G) Growth curves of TbRAP1F/F2H-NLS-ΔMybL (D), TbRAP1F/F2H-ΔNT (E), TbRAP1F/F2H-ΔBRCT (F), and TbRAP1F/ΔMybLΔRCT-F2H-NLSr (G) cells before and after the induction of Cre expression are shown. Download FIG S6, EPS file, 1.9 MB.The Copyright © 2020 Afrin et al.2020Afrin et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the TbRAP1 may also interact with itself (see below). To avoid detecting possible indirect interactions between TbTRF and mutant TbRAP1 mediated by the WT TbRAP1, we induced Cre in these TbRAP1F/F2H-mut strains for 30 h to ensure the depletion of the WT TbRAP1 protein  and in TbRAP1F2H-NLS-ΔMybL/+ TRFi cells, IP experiments were performed using the 12CA5 HA antibody, and the IP products were examined by Western blotting using both the HA probe antibody  of TbRAP1 cannot. Therefore, the RNAi approach is not suitable for studying phenotypes of all domain deletion mutants or point mutations of TbRAP1. In this study, we took advantage of the Cre-loxP system  are essential for cell viability, and its RCT domain, which is important for telomere length regulation and telomeric silencing, is not essential for cell survival  and appropriate antibiotics.All TbRAP1F/+ strain, the NotI/XhoI-digested TbRAP1-5′UTR-loxP-HYGGFPTK plasmid and NotI/XhoI-digested TbRAP1-3′UTR-BSDGFPTK-loxP plasmid were sequentially transfected into SM cells. The A1 and A3 clones of TbRAP1F/+ (-Cre-EP1) were confirmed by Western and Southern analyses (see BLE) marker (TbRAP1F/+ strain. Clones A1 and B1 were verified by their sensitivity to hygromycin and blasticidin after induction of Cre expression by the use of doxycycline (To establish the ) marker  was subsycycline .TbRAP1F/− strain, the NotI/XhoI-digested pSK-TbRAP1-ko-PUR plasmid was transfected into TbRAP1F/+ cells. The pSK-TbRAP1-ko-PUR plasmid contains PUR flanked by sequences upstream and downstream of the TbRAP1 open reading frame (ORF), respectively.To establish the TbRAP1F/F2H-mut strains were established using the same strategy. N-terminal F2H-tagged TbRAP1ΔNT, TbRAP1ΔBRCT, TbRAP1ΔMyb, TbRAP1ΔMybL, and NLS-TbRAP1ΔMybL mutants flanked by sequences upstream and downstream of the TbRAP1 gene, together with a PUR marker, were cloned into pSK to generate respective targeting constructs. The TbRAP1ΔMybLΔRCT-F2H-NLSr mutant flanked by sequences upstream and downstream of the TbRAP1 gene was also cloned into pSK with a PUR marker to generate the mutant targeting construct. All mutant targeting plasmids were digested with SacII (or with PvuII in the case of ΔMybLΔRCT) before transfection of the TbRAP1F/+ cells was performed to generate the corresponding TbRAP1F/F2H-mut strains. All mutant strains were confirmed by Southern analyses.All TbRAP1 self-interaction in the presence and absence of TbTRF, a TbTRF RNAi (TRFi) strain was first established by transfection of the NotI-digested pZJMβ-TbTRF-Mid1 RNAi construct  The importin α C-terminal myc13 tagging fragment was amplified by PCR with primers OBL-TbIMPORT-CT-MYC-FW (5′-GAGGGGGCACCTCAGCAGTTTGAGTTGGGGATGGATCATGGAGATCCAAATGGACAGCCACCACAGGGCCAGTTCGATCTTATCCCCGGGTTAATTAACG-3′) and OBL-TbIMPORT-CT-MYC-BW (5′-GTATATATATATACACGTGCCCTCCTTCTTCACCATTTTCATCCCTTTCATCTCGATCTCATTATATTTATCAACTTTTCTCATCTAGATTCCTTTGCCC-3′) by the use of a plasmid containing myc13 and a HYG marker as the template. The PCR product was then purified and transfected into TbRAP1+/+, TbRAP1F2H+/+ TRFi, and TbRAP1F2H-ΔMybL/+ TRFi cells.Insertion of 13 C-terminal repeats of myc into one endogenous allele of importin α was performed in T. brucei cells in log-phase growth were used for each IP using appropriate antibody or IgG (as a negative control). IP products were pulled down by the use of Dynabeads protein G (Life Technologies) and split equally for two Western blotting analyses using appropriate antibodies. A 1% volume of input sample was loaded as a control. Since F2H-TbRAP1ΔNT, F2H-TbRAP1ΔBRCT, and TbRAP1ΔMybLΔRCT-F2H-NLSr express at low levels, 500 million mutant cells were used for each IP. A 1% volume of input was loaded in Western analysis as a control.A total of 200 million IF experiments were performed as described previously . SpecifiT. brucei cDNA library was prepared by Bio S&T. Briefly, using a modified SMART cDNA synthesis method, the total RNA from WT T. brucei cells was used for synthesis of cDNA with either oligo(dT) or a random primer. The cDNA was normalized and amplified, after which it was inserted into a modified pGAD T7 yeast expression vector.A normalized TbRAP1 MybLike domain was cloned into the pBTM116 vector and transformed into the yeast strain L41 . The resulting cells were transformed with the normalized T. brucei cDNA library. A total of 16.5 million primary transformants were plated onto synthetic drop-out (SD) plates without tryptophan, leucine, or histidine. A total of 711 clones were obtained from this initial screening. Subsequently, these clones were tested by the use of filter lift assays, and 508 candidates were verified to express the reporter lacZ gene. The pGAD T7 candidate plasmids were isolated from these yeast transformants and T. brucei gene insertions were analyzed by restriction digestion, PCR, and sequencing.The TbRAP1F/F2H-ΔMyb and TbRAP1F/+ cells for 30 h, after which total RNA was isolated and purified through RNeasy columns (Qiagen). Three independent inductions were performed as biological replicates. RNA samples were run on a BioAnalyzer 2100 system (Agilent Technologies) using an Agilent RNA 6000 Nano kit to verify the RNA quality and then sent to Novogene for library preparation and RNA high-throughput sequencing.Cre expression was induced by the use of doxycycline in The following processes were performed at Novogene.RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked using a NanoPhotometer spectrophotometer (Implen). RNA integrity and quantitation were assessed using an RNA Nano 6000 assay kit on a Bioanalyzer 2100 system (Agilent Technologies).Sequencing libraries were generated using a NEBNext Ultra RNA library prep kit for Illumina  using 1 μg poly(A) RNA according to the manufacturer’s protocol, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly(T) oligonucleotide-attached magnetic beads. Fragmentation was carried out using divalent cations under conditions of elevated temperature in NEBNext first-strand synthesis reaction buffer (5×). First-strand cDNA was synthesized using random hexamer primers and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RNase H-). Second-strand cDNA synthesis was performed using DNA polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments preferentially of length 150 to 200 bp, the library fragments were purified with an AMPure XP system (Beckman Coulter). A 3-μl volume of USER Enzyme  was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C followed by PCR, which was performed with Phusion high-fidelity DNA polymerase, universal PCR primers, and an Index (X) primer. PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system.Clustering of the index-coded samples was performed on a cBot cluster generation system using a cBot-HiSeq (HS) paired-end (PE) cluster kit (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and 125-bp and 150-bp paired-end reads were generated.The RNA-seq data were analyzed by Novogene as follows.Raw reads of fastq format were first processed through the use of Novogene perl scripts. In this step, clean reads were obtained by removing reads containing adapters, reads containing poly(N), and low-quality reads. At the same time, the levels of Q20, Q30, and GC content of the clean reads were calculated. All downstream analyses were performed on the basis of the clean reads with high quality.T. brucei Lister 427 genome TriTrypDB-45_TbruceiLister427_2018_Genome.fasta and its annotation TriTrypDB-45_TbruceiLister427_2018.gff were downloaded from TriTrypDB and used as the references. The index of the reference genome was built using hisat2 2.1.0, and paired-end clean reads were aligned to the reference genome using HISAT2.The HTSeq v0.6.1 was used to calculate the number of reads mapped to each gene. The number of fragments per kilobase per million (FPKM) was calculated for each gene on the basis of the length of the gene and the number of reads mapped to the gene.P values were adjusted using the Benjamini and Hochberg approach for controlling the false-discovery rate. Genes with an adjusted P value of <0.05 found by DESeq were assigned as differentially expressed.Differential expression analysis of two conditions/group  was performed using the DESeq R package (1.18.0). The DESeq R package provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting GSE143456.The RNA-seq data set determined in this work has been submitted to NCBI GEO under accession number


KIAA1549-BRAF fusions and the H3F3A p.K27M mutation have been described in low-grade (LG) and high-grade (HG) IMAs, respectively. In the present study, we collected clinico-radiological data and performed targeted next-generation sequencing for 61 IMAs  to identify KIAA1549-BRAF fusions and mutations in 33 genes commonly implicated in gliomas and the 1p/19q regions. One hundred seventeen brain astrocytomas were analyzed for comparison. While we did not observe a difference in clinico-radiological features between LG and HG IMAs, we observed significantly different overall survival (OS) and event-free survival (EFS). Multivariate analysis showed that the tumor grade was associated with better OS while EFS was strongly impacted by tumor grade and surgery, with higher rates of disease progression in cases in which only biopsy could be performed. For LG IMAs, EFS was only impacted by surgery and not by grade. The most common mutations found in IMAs involved TP53, H3F3A p.K27M and ATRX. As in the brain, grade I pilocytic IMAs frequently harbored KIAA1549-BRAF fusions but with different fusion types. Non-canonical IDH mutations were observed in only 2 grade II diffuse IMAs. No EGFR or TERT promoter alterations were found in IDH wild-type grade II diffuse IMAs. These latter tumors seem to have a good prognosis, and only 2 cases underwent anaplastic evolution. All of the HG IMAs presented at least one molecular alteration, with the most frequent one being the H3F3A p.K27M mutation. The H3F3A p.K27M mutation showed significant associations with OS and EFS after multivariate analysis. This study emphasizes that IMAs have distinct clinico-radiological, natural evolution and molecular landscapes from brain astrocytomas.Intramedullary astrocytomas (IMAs) are rare tumors, and few studies specific to the molecular alterations of IMAs have been performed. Recently,  BRAF and KIAA1549 genes are found in nearly 80% of cerebellar grade I astrocytomas but only 50–55% of non-cerebellar grade I cases [H3F3A gene (H3K27M) [The recently updated 4th edition of the World Health Organization (WHO) Classification of Central Nervous System (CNS) tumors in 2016 (WHO 2016) drastically changed histological diagnosis in neuropathology by integrating molecular data into daily diagnostic practice , 51. The I cases . Similar(H3K27M)  predomin(H3K27M) .Only a few studies specifically addressing intramedullary gliomas are available. Intramedullary tumors are rare CNS neoplasms accounting for 2 to 4% of all CNS tumors , 48. TheKIAA1549-BRAF and H3K27M mutations [IDH1 p.R132H and IDH2 p.R172H mutations [The few available studies specific to the molecular alterations of intramedullary astrocytomas (IMAs) have been performed and have shown that the most frequent recurrent molecular alterations are fusions involving utations . To dateutations , 47, 54.Nevertheless, while the genetic profiles of brain astrocytomas have been largely established, those of uncommon IMAs remain to be defined. The present study aimed to correlate clinical, radiological and molecular data of IMAs to improve the current knowledge about the prognosis and molecular profile of these tumors.KIAA1549-BRAF fusion gene led to grade I pilocytic astrocytoma diagnosis in cases for which a differential diagnosis between grade I pilocytic and grade II diffuse astrocytomas could not be made [KIAA1549-BRAF fusion” panels (see below). This series included 16 grade I astrocytomas, 13 grade II astrocytomas, 13 grade III astrocytomas and 75 grade IV astrocytomas.Institutional Review Board approval for a retrospective analysis of archival biobank tissue was obtained from Biobanque Hôpital Erasme-ULB (BERA), BE_NBWB1, Biothèque Wallonie Bruxelles (BWB), BBMRI-ERIC) and Biolibrary of Saint-Luc University Hospital, together with ethical agreement. For the present study, the following inclusion criteria were defined: pathological diagnosis of LG or HG astrocytoma and spine location. We excluded cases with a secondary supratentorial location at diagnosis. Pathological diagnosis was reviewed by two neuropathologists (LL and IS) according to the WHO 2016 classification . Accordi be made . Grade In = 34) by an expert neuroradiologist (DB). For the remaining cases, imaging features were collected from MRI reports. The imaging features assessed were location, signal T1 and T2 intensity, contrast enhancement, well-delineated vs. infiltrative pattern, cystic component and necrosis. Figure Magnetic resonance imaging (MRI) exams were reviewed for available cases , adjuvant treatments, follow-up duration and survival status. Based on MRI reports (contrast enhancement and/or tumor size increase), event-free survival (EFS) was assessed as the time until recurrence after gross total resection or disease progression after biopsy/partial resection, as defined by Pekmezci et al.  The mediBriefly, DNA and RNA from formalin-fixed paraffin-embedded (FFPE) tumor tissues were extracted using the QIAamp DNA FFPE Tissue Kit  and the Maxwell RSC DNA FFPE Kit  and were quantified using a Qubit 2.0 Fluorometer . Library construction and quantification were performed using the Ion AmpliSeq Library Kit v2.0 and the Ion Library Quantitation Kit (ThermoFisher Scientific), respectively. Libraries were multiplexed and submitted for emulsion PCR using the Ion Chef System and were sequenced using the Ion GeneStudio S5 System, according to the manufacturer’s instructions (ThermoFisher Scientific).For DNA analysis, two Ampliseq gene-targeted custom panels  were used to analyze a total of 33 genes commonly implicated in gliomas and the 1p and 19q regions  (Supplementary Table To detect https://gnomad.broadinstitute.org/), COSMIC (http://www.sanger.ac.uk/cosmic) and CbioPortal  databases. Variants were classified as ones with pathogenic/potential clinical significance or as variants of unknown significance (VUS) following previous recommendations [For DNA analyses, reads were aligned against the hg19 reference genome with the Torrent Mapping Alignment Program . After alignment, exonic nonsynonymous and splice variants were filtered to exclude those with fewer than 100 reads, present in the population with a minor allele frequency of more than 1% according to the 1000 Genomes project, with mutant allele coverage of fewer than 30 reads or with mutant allele frequency lower than 5% (except for well-known hotspot mutations). The final list of mutations was visualized with the Integrative Genomic Viewer , and systematic sequencing errors or FFPE artifacts were removed. Curated information on variants was obtained from the gnomAD  data were filtered to exclude regions with low confidence, as recommended by the manufacturer (ThermoFisher).Ion reporter software (ThermoFisher) was used for copy number variation detection, including high-level amplification of For RNA analyses, reads were aligned with the hg19 human reference genome, and fusions were identified by using Ion Reporter software v5.10 (ThermoFisher).Statistical analyses were performed using Statistica software .After checking the application conditions, the chi-square test or Fisher’s exact test was used to analyze the associations between categorical variables.p < 0.05.Survival data were subjected to Kaplan-Meier analysis and the log-rank test. Multivariate Cox regression method was also applied. Overall survival (OS) and event-free survival (EFS) were calculated from the initial diagnosis. To include categorical variables in the multivariate models, we created dummy variables as follows. The extent of resection was introduced by means of 2 binary variables: biopsy (no/yes) and total resection (no/yes), with partial resection corresponding to the value of 0 for each of these 2 binary variables. In some cases, the total resection variable could be not included because of the problem of singular matrix decomposition for estimating model parameters. Statistical significance was defined as p > 0.05). However, despite these radiological and clinical similarities, OS and EFS differed widely between patients with LG and HG tumors , whereas a highly significant contribution of grade was confirmed , and total surgical resection did not significantly contribute to better prognosis as an independent factor (p = 0.408) .Analyses of LG IMAs revealed no significant differences between grade I pilocytic and grade II diffuse astrocytomas in terms of age and gender Table . Analysep = 0.011)  between grade I pilocytic and grade II diffuse astrocytomas   for pediatric vs. adult LG patients.EFS was longer in grade I pilocytic astrocytomas  than in grade II diffuse astrocytomas  . However, without distinguishing between grade I and II, there was a statistically significant difference in EFS between biopsy and resected LG cases .Among LG tumors, surgery strongly impacted EFS in both grade I pilocytic and grade II diffuse IMAs (log-rank test: Multivariate Cox regression combining grade (I vs. II) and two surgery variables  revealed that only the absence of surgery  significantly and negatively impacted the EFS of LG astrocytomas, independently of the other variables in the model , 18% (11/61) and 13% (10/61) of cases, respectively. Pathogenic hotspot mutations were identified: BRAF p.V600E mutation , KRAS p.G12V/S and p.Q61R , IDH1 p.R132S , IDH2 p.R172M , ACVR1 p.G328V  and TERT promoter . Other variants were identified in the NF1, PDFGRA, TSC2, TSC1, MSH6, CIC, KEL, PIK3CA, PIK3R1, EGFR, TP73, LZTR1, DAXX, MET, FUBP1 and FOXR2 genes, most of them being VUS. In contrast to brain astrocytomas, no hotspot IDH1 p.R132H or IDH2 p.R172H mutations were identified, and none of our cases harbored the 1p19q codeletion. Regarding the KIAA1549-BRAF fusion, 16% of the contributive cases (10/54) were positive. The different breakpoints identified were KIAA1549(15)-BRAF(9) (8 cases), KIAA1549(16)-BRAF(9) (1 case) and KIAA1549(15)-BRAF(11) (1 case).The molecular findings are summarized in Fig. BRAF alterations (mutations and fusions), KRAS and NF1 mutations were identified in 13, 2 and 1 cases, respectively. For grade I pilocytic IMAs, we observed MAP kinase signaling pathway alterations in 50% of cases (13/26). For the remaining grade I pilocytic IMAs, we did not detect any molecular alteration in 5 cases, while 8 cases presented at least one variant, including mutations of EGFR (one case), PDGFRA (one case), PIK3R1 and the TERT promoter (one case)  and ACVR1 (1 case), and 8 grade II diffuse IMAs did not have any alterations identified with the gene-targeted strategy used here.Alterations in genes involved in the MAP kinase signaling pathway were frequently found in LG IMAs : H3F3A p.K27M mutation, which was found in 73% of cases (11/15) including 11 of 12 grade IV IMAs. Four of the HG IMAs did not harbor the H3F3A p.K27M mutation. Remarkably, the only grade IV IMA that did not carry this mutation was diagnosed in a Lynch syndrome context (case 63) and the others HG IMAs were grade III IMAs. The only alteration identified in 2 of these 3 grade III IMAs cases was the mutation of ATRX. ATRX mutations were identified in only 2 of 11 of the H3K27M-mutant cases (18%), while 3 of 4 HG H3K27M-wild-type IMAs harbored ATRX alterations.All HG IMA cases presented at least one mutation. The most frequent one was the TP53 mutation , which was identified in most H3K27M-mutant tumors . In addition, the majority of NF1 mutations (3 of 4), as well as the only PIK3CA mutation identified, were found in HG IMAs. Importantly, none of the HG cases harbored KIAA1549-BRAF fusions and/or BRAF p.V600E mutations.Another common molecular alteration in HG IMAs was KIAA1549-BRAF fusion” panels revealed that 98.2% (112/114) of brain astrocytomas had at least one molecular alteration (mutation or fusion), while only 65% (36/55) of IMAs had at least one alteration  with the KIAA1549-BRAF fusion being the most common molecular alteration found in both locations. Nevertheless, although the KIAA1549(16)-BRAF(9) fusion was most common in brain grade I pilocytic astrocytomas , the KIAA1549(15)-BRAF(9) fusion was the most common in spinal grade I pilocytic astrocytomas . No other mutations were found in brain grade I pilocytic astrocytomas, while 21% (5/24) of grade I pilocytic IMAs presented mutations in the TP53 or ATRX gene (Fisher test: p = 0.071).At least one molecular alteration was found in 87.5% (14/16) and 71% (17/24) of brain and spinal grade I pilocytic astrocytoma cases, respectively (Fisher test: p = 0.00013). IDH1 p.R132H was the most common mutation found in brain grade II diffuse astrocytomas , while only 2 spine grade II diffuse astrocytomas cases harbored IDH mutations , and these mutations were non-canonical IDH mutations. In both brain and spine tumors, TP53 and ATRX mutations were found concomitantly with IDH mutations. Among IDH-wild-type grade II diffuse astrocytomas, the TERT promoter mutation was the most common mutation found in the brain  compared to BRAF p.V600E  mutation in the spine (Fisher test: p = 0.037).One hundred percent (13/13) of the brain grade II diffuse astrocytomas harbored at least one molecular alteration, compared to only 31.3% (5/16) in the spine , while the H3F3A p.K27M mutation was found in nearly all grade IV IMAs . Moreover, no IDH mutations were found in grade IV astrocytomas located in the spine, while 6.9% (5/72) of the grade IV astrocytomas were mutated in the brain (Fisher test: p = 1.0). Among IDH-wild-type grade IV brain astrocytomas, EGFR alterations (mutation and amplification) and TP53 and PTEN mutations were the most common molecular alterations after TERT promoter mutation. However, in spine grade IV astrocytomas, TP53 and ATRX mutations were the most common ones after H3F3A p.K27M mutation, and no PTEN and TERT promoter mutations were found.For HG astrocytomas, no comparison between grade III astrocytomas of the brain and spine was possible because of the small number of samples from the spine. However, all grade IV astrocytomas, either in the brain or in the spine, harbored at least one mutation. KIAA1549-BRAF fusion was associated with good prognosis; none of the KIAA1549-BRAF-positive cases died, and only 2 of them had recurrence after gross total resection or disease progression after biopsy/partial resection after delays of 28 (case 28) and 115 months (case 10s).Among the 11 H3K27M-mutant IMA cases, 8 of 11 cases died, with a median OS of 20 months, and 7 of 11 showed recurrence after gross total resection or disease progression after biopsy/partial resection with a median EFS of 6 months. In contrast, n = 26), the absence of the KIAA1549-BRAF fusion (n = 16) did not contribute significantly to the prognosis in terms of EFS .Among grade I pilocytic IMAs  IDH mutation (IDH2 p.R172M) (case 21) and the other, an ACVR1 p.G328V mutation (case 50). For the third case (case 55), no molecular alterations could be identified. In the two LG IMAs cases that progressed to a higher grade, one harbored a non-canonical IDH mutation (IDH1 p.R132S) (case 31) and the other a BRAF p.V600E mutation (case 24).Of the 3 LG IMAs cases which died, two harbored n = 15), H3K27M-wild-type cases (n = 4) seemed to have a better prognosis; only 1 case died (median follow-up of 29 months) compared to 8 cases among H3K27M-mutant cases (n = 11) (median follow-up of 20 months). Statistical analyses comparing H3K27M-mutant and H3K27M-wild-type HG IMAs could not be performed due to the small number of H3K27M-wild-type cases.Among HG IMAs  and grade II (40% recurrence) [Nevertheless, even if the histological grade can be challenging to assess in the spine , 41, it urrence) . This ouKIAA1549-BRAF fusions in both locations. However, the most frequent breakpoints found in IMAs were different than those usually found in brain grade I pilocytic astrocytomas, as also described by Faulkner et al. [KIAA1549-BRAF fusion on prognosis remains debated [KIAA1549-BRAF had no impact on either EFS or OS independent of surgery when considering all cases nor on EFS when considering only grade I pilocytic IMAs.IMAs also appear to be molecularly distinct from brain astrocytomas. Grade I pilocytic astrocytomas were characterized by r et al. . The imp debated , with so debated , 24. In IDH1 p.R132S mutation that we found [IDH1 p.R132S-mutated grade II spinal cord astrocytoma [Molecular alterations of grade II diffuse astrocytomas differ according to their location. IDH mutations were found in half of the brain astrocytoma cases, but only in 2 of the 61 IMAs . IDH mutations are generally rare and mostly non-canonical in midline locations . Previouwe found . While frocytoma . This fiEGFR amplification, TERT promoter mutations or combined whole chromosome 7 gain and whole chromosome 10 loss and have glioblastoma-like behavior [IDH1” mutations in pediatric hemispheric diffuse astrocytomas [BRAF p.V600E mutation, FGFR1 alterations or MYB or MYBL1 rearrangement [EGFR or TERT promoter mutations. We observed BRAF p.V600E in 2 cases and found no other specific molecular alteration except for one hotspot ACVR1 p. G328V mutation, mostly described in “diffuse intrinsic pontine glioma” (DIPG) [FGFR1, MYB and MYBL1 were not covered by the gene-targeted NGS used in the present study. Recent studies have reported that chromosomal rearrangements in gliomas resulting in transcript fusion gene, such as NTRK 1/2/3 or FGFR1/2/3 fusions, could be therapeutic targets [FGFR inhibitors showed promising effects as targeted treatments for gliomas [As described recently, “adult-type” grade II diffuse astrocytomas with no IDH mutations often harbor behavior . In contocytomas , and theocytomas . The molangement , 37. In ” (DIPG)  and in o targets  and are  targets  or Larot targets ). Clinic gliomas , 18. BecIn the brain, the natural evolution of most “adult-type” grade II diffuse gliomas is to progress to a higher grade , 37. AnaH3F3A mutations identified in our cohort consisted of the recurrent hotspot p.K27M mutation. We did not find any other mutations in genes encoding histone variants H3.1 or additional H3F3A mutations, in contrast with the recent report by Sloan et al., who reported H3F3A p.G34W in grade II and III spinal cord astrocytomas [TERT promoter mutations among HG IMAs, in contrast with those in the brain, and with two recent studies [TERT promoter mutations and H3K27M mutations [TERT promoter mutations, and the only alteration identified was the ATRX mutation in two of them.In this cohort, nearly all grade IV IMAs were H3K27M mutants (92%), in line with previous reports, which described a range between 38 and 100% of H3K27M-mutant tumors among HG spinal astrocytomas , 44, 53.ocytomas .We did nutations . InteresKIAA1549-BRAF fusion and H3F3A p.K27M mutations were identified in our cohort, in agreement with Shankar et al. [, who did not report any recurrent molecular alterations [No recurrent molecular alterations other than r et al.  and the EGFR and TERT promoter mutations, associated with diagnosis and prognosis in the brain, do not seem to occur with the same frequency, have the same implications in spine or at least, deserve further study. In the brain, assessment of IDH mutations has been shown, as Ellezam et al. reported, to help to distinguish grade I pilocytic, typically IDH wild-type, from grade II diffuse astrocytomas [IDH1 p.R132H in IMAs.In conclusion, these specific clinico-radiological and molecular landscapes of IMAs suggest that diagnostic algorithms commonly used in the brain must be reviewed to be confirmed as appropriate for those in the spine. First, assessment of the imaging infiltrative/well-delineated pattern, as commonly used for the differential diagnosis of grade I pilocytic astrocytomas and diffuse grade II, III and IV astrocytomas, seems not to be useful in the spine. Moreover, classical molecular alterations such as IDH mutations, ocytomas . In the H3F3A p.K27M mutation is an important molecular alteration to assess because of its prognostic implications.In addition, the prognostic implications of molecular alterations in IMAs must be well characterized since implications similar to those observed in the brain cannot be established. LG IMAs seem to have a clinical and a molecular profile more related to diffuse “pediatric-type” gliomas than those classically observed in the brain. HG IMAs are strongly related to diffuse midline H3K27M mutant gliomas, and the Additional file 1: Table S1. List of genes, copy number variations (CNV) and KIAA1549-BRAF fusions tested.Additional file 2: Table S2. Prognosis Model for all cases.Additional file 3: Table S3. Event-Free Survival (EFS) Prognosis Model for LG cases (grade I and grade II only).Additional file 4: Table S4. Prognosis Model including molecular data for all cases.Additional file 5: Table S5. Event-free Survival (EFS) Prognosis Model including molecular data for grade I pilocytic and grade II diffuse intramedullary astrocytomas.