Despite S13 and V1 being antigenically similar, only S13 protected ferrets from wt virus shedding and fever post-challenge. Crucially, these data suggested that enhanced fitness allowed S13 to overcome inter-strain competition in quadrivalent LAIV (QLAIV). This improved efficacy was later validated by real-world VE data. S13 displayed increased binding avidity to a mammalian-like α-2,6 receptor analogue (6-SLN), relative to V1, while maintaining avian-like 3-SLN avidity. In silico modelling of the HA receptor binding site revealed additional interactions in the S13:6-SLN binding network and a mild increase in 6-SLN binding energy, indicating a possible mechanism for increased α-2,6 receptor-binding avidity. These data confirm that rational HA mutagenesis can be used to optimise hNEC replication and VE for A/H1N1pdm09 LAIV viruses.During 2013–14 and 2015–16, A/H1N1pdm09 live attenuated influenza vaccine (LAIV) viruses replicated inefficiently in primary human nasal epithelial cells (hNEC). This led to reduced vaccine effectiveness (VE) in quadrivalent formulations, mediated by inter-strain competition. By mutating the haemagglutinin (HA) protein, we aimed to enhance hNEC replication of a novel A/H1N1pdm09 vaccine strain to overcome competition and improve VE. Combinations of N125D, D127E, D222G and R223Q substitutions were introduced to the HA protein of A/Slovenia/2903/2015 (A/SLOV15). A/SLOV15 S13, containing all four HA substitutions, produced approximately 1000-fold more virus than parental V1 during hNEC infection. Immunogenicity in ferrets was increased by approximately 10-fold, without compromising yield in eggs or antigenic match to wild-type ( Vaccine effectiveness for the pandemic H1N1 component of the FluMist/Fluenz live attenuated influenza vaccine was lower than expected during the 2013–14 and 2015–16 influenza seasons in the USA. Previous studies highlighted the importance of vaccine viruses replicating sufficiently well in human cells that they could compete in quadrivalent formulations and protect ferrets against influenza-like illness.Here, it was demonstrated that mutagenesis of the haemagglutinin surface protein can be used to improve vaccine effectiveness of a pandemic H1N1 vaccine strain. This was achieved by optimising replication in human cells while, crucially, leaving other key characteristics required for vaccine production unaffected. The mutated vaccine virus successfully overcame inter-strain competition, providing protection from influenza-like illness in ferrets, while its parent did not. Furthermore, it was demonstrated that enhanced binding to human-like receptors was the mechanism by which replicative fitness was being improved.These data suggested that improving replicative fitness, allowing the vaccine virus to drive a protective immune response in quadrivalent formulations, would result in increased effectiveness in patients. Subsequently, high vaccine effectiveness was demonstrated during the 2017–18 northern hemisphere influenza season, validating this approach. Consequr/6/1960 –9 for A-r/6/1960 –12, to pr/6/1960 ,14.in vivo using an optimised ferret challenge model, which demonstrated that the reduced fitness of A/BOL13 led to inter-strain competition and loss of efficacy in multivalent vaccine formulations [During the 2013–14 and 2015–16 influenza seasons, the A/H1N1pdm09 components of QLAIV, A/California/07/2009 (A/CA09) and A/Bolivia/559/2013 (A/BOL13), respectively, were found to have reduced vaccine effectiveness (VE) in the USA . A/H1N1pulations .To improve on the reduced VE of A/BOL13, the A/H1N1pdm09 component of FluMist/Fluenz required updating to a strain with enhanced replicative fitness in hNEC for the 2017–18 season. To achieve this, the use of rational mutagenesis of the HA protein was explored.While HA protein mutagenesis had been used successfully to enhance egg yield in the past ,21, optiwt strains carrying the D222G substitution were also associated with increased disease severity during the 2009 influenza pandemic [A representative of the newly emerged 6B.1 clade, A/Slovenia/2903/2015 (A/SLOV15), was chosen to undergo HA mutagenesis to optimise replicative fitness in hNEC. Four amino acid substitutions were selected for introduction into the HA protein of A/SLOV15: N125D, D127E, D222G and R223Q. These substitutions were initially identified in LAIV as minor quasispecies during vaccine development of egg-grown A/H1N1pdm09 strains ,25. The pandemic . RestoraHere we used HA mutagenesis to optimise the replication of A/SLOV15 in hNEC, while maintaining sufficient egg yield for LAIV manufacture and without compromising virus immunogenicity or antigenicity. We aimed to demonstrate that by optimising replicative fitness in human cells, A/SLOV15 would overcome the inter-strain interference observed for A/BOL13  and provwt A/SLOV15 HA protein to generate a panel of A/SLOV15 LAIV mutants carrying between 1–5 amino acid changes relative to wt (50)/cell and collecting apical washes daily during a five day time-course. Samples were taken from nine transwells per virus, across 3 independent experiments and titrated by TCID50. Replication of A/SLOV15 LAIV viruses in hNEC was expressed as the mean daily virus production from each hNEC transwell over five days of infection, as a measure of each mutant’s ability to establish sustained virus replication. This generated a single data point per transwell for statistical analysis, while still utilising all time-course data as described previously [Site directed mutagenesis was performed on the egg-derived ve to wt . N125D, eviously ,32  also replicated to higher titres in hNEC than V1, with a mean virus titre of 4.75 Log10TCID50/mL per day over five days, but this was not statistically significant , wt A/SLOV15, A/SLOV15 V1, A/SLOV15 S2-S5 single mutants and A/SLOV15 S13. A/MICH15 wt was included as the WHO-recommended A/H1N1pdm09 strain during the 2017–18 season along with the A/SLOV15 parental wt. Single mutants S2-S5 were included to assess individual contributions of each mutation to antigenicity and immunogenicity profiles relative to the wt reference strains and V1. S13 was also included in this assay due to replicating to the highest titres in hNEC and eggs. Results were expressed as geometric mean of HAI titres obtained from the three independent HAI assays.To understand any impact of HA mutagenesis on the antigenic profile of A/SLOV15, serum immune responses and antigenic cross-reactivity of serum antibodies were assessed by two-way haemagglutination inhibition (HAI) assay. Ferret antisera was raised by vaccinating one ferret per Animals that received A/SLOV15 S2 and S4 generated comparable serum antibody titres relative to A/SLOV15 V1, showing that the N125D and D222G single mutations had minimal measurable impact on serum antibody response by HAI . Howeverwt A/SLOV15 and wt A/MICH15 were able to inhibit haemagglutination by all LAIV variants and both reference wt viruses. In all cases HAI titres were within four-fold of the relevant homologous titre and therefore considered antigenically similar. Vaccination with A/SLOV15 S3 did not produce a detectable serum antibody response in ferrets, meaning that antigenic cross-reactivity could not be determined for antisera from this variant. However, antisera raised against the other LAIV and wt viruses in this experiment were all able to recognise A/SLOV15 S3 LAIV virus within four-fold of homologous titre.HA mutagenesis had little impact on the antigenic profiles of the A/SLOV15 LAIV variants included in this study when assessed by two-way HAI. Despite differences in immune responses generated, antisera raised against A/SLOV15 V1, S2, S4, S5 and S13, wt A/MICH15 and wt A/SLOV15 were used to examine the antigenic profiles of all A/SLOV15 LAIV variants in a one-way HAI . This indicated that a sufficient level of hNEC replication was required to generate serum immune responses in ferrets in this low dose model. Given their comparable serum antibody titres, S13 was selected for further characterisation due to its superior fitness in hNEC.Animals that received a low dose of A/SLOV15 V1 and S3 failed to generate a serum immune response that was detectable by HAI, and were identical to the unvaccinated group . In contReduced thermal stability of A/CA09 HA protein may have contributed to lower than expected VE estimates for this strain –24 durinThis accelerated thermostability assay showed that A/SLOV15 V1 and A/CA09 both lost the ability to agglutinate red blood cells at 55°C, indicating that A/SLOV15 V1 was similarly thermally unstable to A/CA09. However, HA thermostability of A/SLOV15 S13 was improved relative to A/SLOV15 V1, with this variant losing the ability to agglutinate red blood cells at 60°C. While not as stable as A/NC99, with a ‘shut-off’ temperature of 65°C, this indicated that the five amino acid changes present in S13 , resulte10 FFU of the appropriate formulation. Vaccine formulations included in this study were monovalent A/SLOV15 V1 (MLAIV-V1), monovalent A/SLOV15 S13 (MLAIV-S13), A/SLOV15 V1 in a representative 2017–18 quadrivalent formulation (QLAIV-V1), A/SLOV15 S13 in a representative 2017–18 quadrivalent formulation (QLAIV-S13) and an unvaccinated group. Nasal swabs were collected daily for five days post-vaccination to measure monovalent LAIV shedding. Bleeds were collected 21 days post-vaccination to assess serum immune responses. Ferrets were then challenged with 5.0 Log10 FFU wt A/SLOV15 to assess wt shedding and development of fever as measures of protection from influenza-like illness (ILI). Nasal swab samples were collected for three days post-challenge to measure wt shedding and core body temperature data obtained from the implanted telemetry chips allowed assessment of fever . One animal in the QLAIV-S13 group failed to produce a serum antibody response, while the three remaining animals generated titres of 5–7 log2, comparable to MLAIV-S13. However, due to the spread of the data this improvement was not statistically significant.Serum immune responses were assessed 21 days post-vaccination. There were no detectable antibody titres for any animals in the MLAIV-V1 and QLAIV-V1 groups when measured by HAI . Converswt A/SLOV15 on day 28 post-vaccination, to assess relative levels of protection provided by V1 and S13. Shedding of wt virus was measured by collecting nasal swabs daily for three days post-challenge and measuring infectious virus shedding by TCID50. A single data point per animal was obtained for statistical comparison by calculating the geometric mean of TCID50 titres obtained for the three days . Two animals in the QLAIV-S13 group failed to reduce wt virus titre, but the two remaining animals produced wt shedding titres approaching the limit of detection of the assay. This indicated that two out of four ferrets vaccinated with QLAIV-S13 were protected from wt challenge . Development of fever was prevented in three out of four animals in the QLAIV-S13 group, which was a statistically significant improvement relative to unvaccinated ferrets (P < 0.001).To confirm and strengthen our observations of QLAIV-S13 providing protection from y set up  Fig 5A)wt challewt shedding and fever. However, data obtained from these studies indicated that S13 was of superior replicative fitness and was able to generate robust serum immune responses in vivo in animals vaccinated with both MLAIV and QLAIV. S13 also provided improved protection from ILI in ferrets vaccinated with S13 in monovalent and quadrivalent formulations, relative to V1 and unvaccinated animals.Taken together, these data suggested that A/SLOV15 V1 would not provide protection from ILI due to being unable to generate a serum immune response or prevent 10 HA copies for use in these assays. Binding curves and half-saturation values were then obtained using the Hill equation to apply a non-linear least squares fit to the values obtained for fractional saturation as a function of sugar loading  receptor binding assays on the Octet RED96 instrument. This was to determine the binding avidity of A/SLOV15 S13 relative to A/SLOV15 V1 for avian-like α-2,3 receptor analogues (3-SLN) and human-like α-2,6 receptor analogues (6-SLN). Biotinylated receptor analogues were loaded onto streptavidin biosensors at concentrations of 0.01–0.5μg/mL. Whole viruses were quantified by digital PCR (dPCR) to allow normalisation by HA vRNA content to 6.8 Log loading . OseltamBinding data showed that A/SLOV15 S13 had slightly decreased avidity for 3-SLN relative to A/SLOV15 V1, with half saturation being reached at concentrations of 0.2 μg/mL for A/SLOV15 S13 and 0.08 μg/mL for A/SLOV15 V1 . This inin vitro BLI data, binding energies of the A/SLOV15 V1 and A/SLOV15 S13 HA proteins in complex with the 3-SLN and 6-SLN receptor analogues were estimated in silico by the molecular mechanics with generalised Born and surface area solvation (MM/GBSA) method. A/SLOV15 V1 and S13 sequences were mapped to the A/California/04/2009 crystal structure (PDB ID: 3UBE) and the 3-SLN and 6-SLN sugars were docked to measure binding energies. 3-SLN binding energies were similar for both variants  relative to S12 (day 4) could be important in overcoming inter-strain competition in QLAIV. Conversely, a mutant such as S5, which did reach a peak titre of 5.97 Loge-course . While awt viruses [wt viruses circulating in the human population. Further hNEC experiments using a panel of mutated wt viruses would be required to confirm this.The D222G and R223Q residue changes, located in the RBS, likely contributed to a change in receptor binding specificity for A/SLOV15, from a preference for α-2,3 receptors to dual α-2,3 and α-2,6 specificity ,27–29,33 viruses ,35. It iin vitro and in vivo data during vaccine strain development.In contrast to the hNEC data, introduction of a D127E single mutation resulted in no detectable post-vaccination serum immune response in ferrets as measured by HAI , while Vwt influenza A virus replication in the respiratory tract of ferrets [While hNEC fitness did not correlate with immunogenicity for mutant S3, it correlated well for the remaining A/SLOV15 LAIV viruses tested. More robust serum immune responses were generated in animals vaccinated with S5 and S13, relative to V1 . This wa ferrets . Having 10 FFU vaccine dose, these data suggested that immunogenicity was relatively binary. A/SLOV15 V1 and S3, with lower hNEC fitness, failed to produce a detectable response in any animals in these study groups, while fitter viruses, S8 and S13, generated comparable serum antibody titres, despite significant differences in hNEC replicative fitness. This supported a minimum level of hNEC fitness being required for immunogenicity, but also that serum antibody titres alone might not effectively differentiate between immunogenic strains.Measuring serum immune responses for a cross-section of A/SLOV15 mutants with varying replicative fitness in hNEC indicated that a threshold of viral fitness was required to generate serum antibodies in ferrets. Interestingly, using a more clinically translatable 4 Loget al to investigate the low VE estimates of the A/H1N1pdm09 strain, A/BOL13 [wt shedding and ILI in vivo in monovalent formulation. However, protection was significantly reduced in trivalent and quadrivalent formulations due to competition with other sub-types [Ferret efficacy studies using low vaccine doses were previously conducted by Dibben  A/BOL13 . The stuub-types . This lowt virus challenge at this limited dose wt virus wt challenge  were associated with α-2,3 receptor specificity, while D222G and R223Q (S13) substitutions conferred dual α-2,3 and α-2,6 receptor specificity [A vast improvement in 6-SLN binding avidity was observed for S13 relative to V1, for which there was no detectable binding to the α-2,6 receptor analogue . This sucificity ,27–29. Win silico modelling of receptor binding networks, which suggested the presence of additional interactions between 6SLN and S13: an H-bond with residue 180H and a water bridge formed by 187D, via one of the 6-SLN hydroxyl groups ; the most relevant A/H1N1pdm09 structure available when this work was conducted.When binding energies between individual HA monomers and receptor analogues were predicted by MM/GBSA, a small improvement in 6-SLN affinity was observed for S13, relative to V1 . This wal groups . Due to While increased α-2,6 receptor binding provides a probable mechanism for the enhanced hNEC replication of S13, the contribution of the 125/127 locus remains unclear, particularly the role of the D127E single mutation. These residues are located in close proximity to the RBS, but do not appear to be involved in 6-SLN binding . HoweverOther relevant considerations for LAIV CVV development include egg titre, antigenicity and HA thermostability. Perhaps surprisingly, altering residues within, and in close proximity to, the RBS to improve hNEC replication did not generally compromise peak virus titres in eggs , contrarwt A/SLOV15 and wt A/MICH15  provided ethical approval for the ferret studies detailed below that were carried out at Charles River Laboratories (CRL) Ireland Ltd, Carrentrila, Ballina, Co. Mayo, Ireland, F26 D786. Work was conducted in compliance with the European Directive for the Protection of Animals used for Scientific Purposes, Directive 2010\63\EU, as transposed into Irish law under Statutory Instrument, S.I. No. 543 of 2012 (as amended) and under the HPRA project authorisation to carry out influenza testing in ferrets that is held by CRL (AE19108/P022). The project manager holds a valid individual authorisation from the HPRA (No. 19108\I079) and all staff were trained and hold valid HPRA individual authorisations for performing the procedures and euthanasia required by the studies.Madin-Darby canine kidney (MDCK) cells and HEK-293T cells were cultured and maintained in Eagle’s Minimum Essential Medium (EMEM)  containing non-essential amino acids, sodium pyruvate and supplemented with 10% heat-inactivated fetal bovine serum (v/v) (FBS) , 1% penicillin-streptomycin (v/v) , and 1% 200mM L-Glutamine (v/v) .2 for seven days prior to use, and basal media was replaced every 3–4 days.Fully differentiated hNEC were purchased from Epithelix  and maintained in transwells at the air-liquid interface in serum-free MucilAir culture media (Epithelix), supplemented with manufacturer recommended growth factors. Cells were incubated at 37°C and 5% COSpecific pathogen free embryonated hen’s eggs were obtained from Charles River Laboratories, Wilmington, USA. Embryonated hen’s eggs were incubated at 37°C with rotation and 70% humidity for 10–11 days prior to being inoculated.wt A/SLOV15 virus was provided by the Worldwide Influenza centre at the Francis Crick Institute, UK. The HA and NA sequences of wt A/SLOV15 are available on GISAID (Isolate ID: EPI_ISL_223353). A/SLOV15 HA and NA genes were amplified by RT-PCR and cloned into the pAD3000 vector as previously described [HA gene segment, the wt A/SLOV15 NA gene and six internal gene segments  from the ca, ts, att A/Ann Arbor/6/1960 master donor virus (MDV). A/SLOV15 LAIV mutants were propagated in the allantoic cavity of 10 to 11-day old embryonated hen’s eggs at 33°C and 70% humidity.Egg-grown escribed ,20. Siteescribed ,20. Eachwt A/SLOV15 NA and there were no compensatory mutations detected in HA or NA for all mutants included in this study. A/SLOV15 stocks were also titrated by TCID50 assay as described below.HA and NA sequences of A/SLOV15 viruses were verified by extracting RNA using QIAamp Viral RNA Mini Kit  as per manufacturer’s instructions. The viral RNA was amplified by RT-PCR and the resulting cDNAs underwent Sanger Sequencing. Sanger sequencing confirmed that HA sequences were as described in 50/cell and assuming 500,000 cells per transwell. Prior to infection, the apical surface was washed with phosphate-buffered saline (PBS) pH 7.4  to remove accumulated mucous. 200μL of PBS was applied to the apical surface and cells were incubated at 37°C and 5% CO2 for 20 minutes. Apical supernatant was removed and cells were washed a further seven times with PBS. LAIV viruses were diluted to an MOI of 0.01 in PBS and 50μL/transwell was used to infect hNEC for 1 hour at 33°C and 5% CO2. Inocula were aspirated and cells were washed once with PBS to remove unbound virus. hNECs were incubated at 33°C and 5% CO2 for five days. Virus time-course samples were collected every 24 hours by adding 200μL PBS to the apical surface of each transwell and incubating for 30 minutes at 33°C and 5% CO2. Virus-containing apical supernatants were stored at -80°C until subsequent titration by TCID50 in MDCK cells, as described below.Primary hNEC were inoculated with A/SLOV15 LAIV viruses at an MOI of 0.01 TCID50/egg. Eggs were incubated at 33°C with 70% humidity until the appropriate time-point . Eggs were chilled at 4°C for a minimum of 5 hours prior to allantoic fluid being harvested and pooled within each virus group and timepoint. Time-course samples were titrated by TCID50, as described below, to measure replicative fitness in eggs.A/SLOV15 LAIV mutants were diluted in PBS and 10–11 day-old embryonated hen’s eggs were inoculated with 125 TCIDHA thermostability was determined by measuring the temperature at which test viruses were no longer able to agglutinate red blood cells (shut-off temperature). A/SLOV15 V1, A/SLOV15 S13, A/CA09 and A/NC99 were diluted to 512 HA units and incubated at 47.5°C, 50°C, 52.5°C, 55°C, 57.5°C, 60°C, 62.5°C and 65°C for 20 minutes. After incubation, HA assays were performed according to standard methods using 0.5% chicken red blood cells .Studies were conducted in outbred, mixed sex, specific pathogen free ferrets . Ferrets in the immunogenicity study were 23–30 weeks old, and ferrets in the efficacy study were 14–26 weeks old. Ferrets in both studies underwent daily health checks during the acclimatisation period, which was a minimum of seven days for healthy animals, prior to the study start dates. Once acclimatised, animals in both studies were confirmed seronegative for circulating A/H1N1pdm09 viruses by HAI assay, before being randomly assigned into groups for vaccination and housed in pairs. Once vaccinated, any adverse health observations were reported and investigated by the designated veterinarian.10TCID50/dose (0.1mL per nostril).On study day 0, two ferrets per vaccination group were lightly sedated with isoflurane  and inoculated intranasally with 0.2mL of the appropriate A/SLOV15 LAIV virus at 6.0–7.0 LogOn day 14 post-vaccination, ferrets were euthanized by intracardiac injection of 1.5 ml of sodium pentobarbitone  and blood samples were collected for analysis of serum immune responses. 45 – 90mL blood was collected per animal and allowed to coagulate before centrifuging. Serum was then decanted into 50mL falcon tubes for storage at -80°C.2 for 18-20h in the absence of trypsin. Plates were then fixed with 80% acetone  in water, and stained with subtype specific anti-HA protein primary antibodies followed by fluorescent secondary antibodies. Primary antibodies used for each subtype were as follows: A/H1N1pdm09- sheep anti-A/Michigan/45/2015 , A/H3N2- sheep anti-A/Hong Kong/4801/2014 , B-Vic- B/BRIS08- mouse anti-B/Victoria, C14975 (AstraZeneca), B-Yam- B/PHUK13- mouse anti-B/Yamagata, SP12-051 (AstraZeneca). Fluorescent secondary antibodies used for this assay were as follows: A strains- alexa 488 donkey anti-sheep IgG (H&L) , B strains- alexa 488 goat anti-mouse . Fluorescent foci were then counted using an Eclipse Ti inverted fluorescent microscope (Nikon) at x100 magnification and vaccine virus concentration was calculated and expressed as Log10 FFU/dose.Monovalent and quadrivalent LAIV formulations for use in ferret efficacy studies were titrated by fluorescent focus assay (FFA), to allow measurement of multiple subtypes with strain-specific antibodies, as previously described . BrieflyFerret efficacy studies were performed as described previously . Intrape10FFU/strain of the appropriate A/SLOV15 LAIV formulation or a mock vaccination with the sample diluent . A/SLOV15 LAIV formulations were monovalent A/SLOV15 V1 (MLAIV-V1), quadrivalent A/SLOV15 V1 (QLAIV-V1), monovalent A/SLOV15 S13 (MLAIV-S13) and quadrivalent A/SLOV15 S13 (QLAIV-S13). Quadrivalent formulations consisted of the relevant A/SLOV15 LAIV variant with representative A/H3N2 , B-Victoria (B/Brisbane/60/2008) and B-Yamagata (B/Phuket/3073/2013) strains from the 2017–18 influenza season to simulate the FluMist/Fluenz vaccine.On day 0 of the study, four ferrets per group were lightly sedated with isoflurane and intranasally vaccinated with 4.0 LogNasal swab samples were collected daily on study days 1–5 by anaesthetising the animals with an intramuscular injection of 0.1 mg/kg Medetor  and then sedating with isoflurane. A Copan FloqSwab ) kit 1mL  was inserted and rotated in the right nostril, then eluted by light vortexing in 1mL of Copan universal transport medium before being aliquoted and stored at –80°C until subsequent measurement of virus shedding. The anaesthetic was then reversed with an intramuscular injection of 0.1 mg/kg Revertor , a minimum of 30 minutes after the sedative.10FFU/dose of A/SLOV15 cell wt challenge virus as a 0.2mL dose. Nasal swab samples were collected daily, as described above, for 3 days post wt challenge to allow subsequent measurement of wt shedding by TCID50 assay as described below. Ferrets were euthanised three days post-challenge by intracardiac injection of 1.5 ml of sodium pentobarbitone.2mL bleeds were collected from the superior vena cava on days 21 post-vaccination for the analysis of serum immune responses. On day 28 post-vaccination, ferrets were lightly sedated with isoflurane as above, before being inoculated with 5.0 Logwt viruses). Two-fold dilution series of serum was then performed and 8 HAU of virus added to each well. Plates were incubated at room temperature for 30–40 minutes before addition of 0.5% chicken red blood cell suspension . Plates were incubated for a further 60 minutes and HAI titres recorded as the reciprocal of the highest dilution of antiserum able to fully prevent agglutination.HAI assays were performed by standard methods. 100μL ferret antiserum was combined with 150μL 2x receptor destroying enzyme  and incubated at 37°C for 18–20 hours. 150μL 2% (w/v) sodium citrate was then added followed by heat inactivation at 56°C for 45 minutes. Treated antiserum was diluted in PBS if necessary . Briefly, RNA was extracted from 100μL sucrose purified LAIV virus which made up to a total volume of 500μL with water before being combined with 500μL of RLT buffer and 70% ethanol. The remainder of steps were completed according to RNeasy Mini Kit  manufacturer’s instructions. Final RNA was eluted in 50μL of water.Superscript III First Strand Synthesis System  was used to generate cDNA by following the manufacturer’s protocol. 1μL gene specific HA primer, H1_HA_742F (10μM) (GTAAAACGACGGCCAGTGAGGGGTCAAGAAGGG), which added an M13 tag to cDNA, was combined with 1μL of extracted LAIV RNA.−1–1 x 10−6) to identify the dilution within ‘digital’ quantification range. cDNA was combined with 200nM of the M13 forward primer (GTAAAACGACGGCCAG), 800nM of the gene specific HA reverse primer (GAAATGGGAGGCTGGTG), 100nM of the gene specific hydrolysis probe (FAM/GGACACTAG/ZEN/TAGAGCCGGGA/BHQ), 7.25μL QuantStudio 3D Digital PCR Master Mix V2  and made up to the final volume of 14.5μL with water.14.5μL dPCR reactions were then set up using 5μL cDNA at a range of 10-fold dilutions . Thermal cycling of chips was then performed as follows: 96°C for 10 minutes, 39 cycles of 56°C for 2 minutes, 98°C for 30 seconds and a final extension at 56°C for 2 minutes. Chips were read on the QuantStudio 3D Digital PCR instrument.rd standard deviation of a no template control (NTC) chip. This threshold was applied to all the wells on each chip using the dPCR Analysis Suite Application (Thermo Fisher) and FAM positive and negative well counts were extracted. Copy numbers for HA vRNA were calculated using Poisson statistic formula described previously [Data were analysed using the dPCR Analysis Suite Application (Thermo Fisher). Briefly, the threshold for a positive well on the FAM channel was set by calculating the upper 3eviously .10 HA copies/well in the same buffer. Association was measured for a duration of 30 minutes at 25°C. Blivion software  was used to plot equilibrium measurements of virus binding as a function of the concentration of sugar immobilised on the biosensor calculated from the response during the sugar loading step.Polyacrylamide-linked biotinylated receptor analogues containing 20% mol sugar and 5% mol biotin, α-2,3 sialyl lactosamine (3-SLN) and α-2,6-linked sialyl lactosamine (6-SLN) were purchased from GlycoNZ . Virus binding was measured on the Octet RED96 (ForteBio) by loading 3-SLN and 6-SLN onto streptavidin coated biosensors  at 0.01–0.5μg/mL for 4 minutes in HBS-EP buffer  with 25 μM oseltamivir acid  and 100μM zanamivir , and then virus was added at 6.8 LogStatistical analyses were performed in GraphPad Prism 9.0 . Half saturation measurements were compared by paired t-test. A/SLOV15 V1–6-SLN binding was allocated a half saturation value of 0.5μg/mL, the maximum sugar loading concentration for this assay, due to no detectable binding during these experiments.The crystal structure of the A/H1N1pdm09 strain A/California/04/2009 HA (PDB ID: 3UBE)  was usedMolecular dynamics simulations were run in Desmond, via Schroedinger suite  . The OPSPrime MM/GBSA analysis calculations were run to estimate the binding energy of the sialic acid ,52,58, aData were tested for normal distribution by performing a Shapiro-Wilk test. Subsequent analyses are described in the figure legends. All graphs were generated in GraphPad Prism, version 9.0. Statistical analyses were performed in GraphPad Prism version 9.0.S1 Fig50 assay. Mean virus titre reached at each timepoint across three independent experiments  are shown for A/SLOV15 V1 and (A) A/SLOV15 single mutants; (B) A/SLOV15 double mutants and (C) A/SLOV15 mutants carrying 3–5 substitutions. Error bars indicate standard deviation.Five day time-course infections in hNEC were performed with all A/SLOV15 mutants an MOI of 0.01. Apical wash samples were collected every 24 hours and virus titre measured by TCID(TIF)Click here for additional data file.S2 Fig50/egg of each A/SLOV15 mutant. Virus yield was measured by TCID50 assay of allantoic fluid collected 24h, 48h, 72h and 90h post-infection. Mean virus titre reached at each timepoint across three independent experiments are shown for A/SLOV15 V1 and (A) A/SLOV15 single mutants; (B) A/SLOV15 double mutants and (C) A/SLOV15 mutants carrying 3–5 substitutions. Error bars indicate standard deviation.Embryonated hens eggs were inoculated with 125 TCID(TIF)Click here for additional data file.S3 Figwt virus was measured on days 1–3 post challenge by TCID50. Graphs show wt virus titre measured for the four animals per study group per day, each curve represents an individual animal. Mean wt virus titres obtained from four unvaccinated control animals are shown with wt titres obtained from ferrets vaccinated with (A) MLAIV-V1; (B) QLAIV-V1; (C) MLAIV-S13 and (D) QLAIV-S13. Dotted line indicates lower limit of detection of the TCID50 assay. Values below the LLD are plotted as ½ LLD for statistical purposes.Shedding of (TIF)Click here for additional data file.S4 Figwt challenge, with each temperature curve representing an individual animal. Core body temperature readings for animals vaccinated with (A) MLAIV-V1; (B) MLAIV-S13; (C) QLAIV-V1 and; (D) QLAIV-S13. (E) Core body temperatures of the unvaccinated control group during the three days post-challenge.Ferret core body temperatures were recorded hourly by intraperitoneal chips from pre-vaccination to study termination. Graphs show spline fits to the hourly temperature readings on days 1–3 post-(TIF)Click here for additional data file.S5 Fig10 FFU of either monovalent S13 (MLAIV-S13) or S13 in a representative 2017–18 quadrivalent formulation (QLAIV-S13). Eight unvaccinated ferrets were also included as controls. (A) LAIV shedding was measured by TCID50 using nasal swab samples that were collected daily days 1–5 post vaccination. (B) Serum immune responses were measured by HAI using antisera collected 21 days post-vaccination. MLAIV-S13 and QLAIV-S13 were compared to the unvaccinated control animals by performing a Kruskal-Wallis test followed by Dunn’s multiple comparisons test. (C) wt shedding was measured by TCID50 using nasal swab samples that were collected daily between days 1–3 post-wt challenge and the geometric mean of wt shedding per day for each ferret depicted by the scatter plot. Shedding of vaccinated animals relative to unvaccinated animals was compared by a Kruskal-Wallis test followed by Dunn’s multiple comparison test. (D) Ferret core body temperature was measured hourly from pre-vaccination to study termination. Fever values as a measure of influenza-like illness for individual ferrets were obtained by subtracting temperature values recorded post-wt challenge from baseline temperature for each animal. Each vaccinated group was compared to the unvaccinated control group by one-way ANOVA followed by Dunnet’s multiple comparisons test. Mean values for individual animals are depicted by the scatter plots (B-D), mean values for all animals in each vaccination group are indicated by the columns (B-D), error bars show standard deviation  or median with 95% CI , and the dotted lines represent the lower limit of detection (LLD) for each assay (B-D). Values below the LLD are plotted as ½ LLD for statistical purposes. P values are indicated as follows: **** P<0.0001, *** P<0.001, ** P<0.01 and * P<0.05 (B-E).Four ferrets were vaccinated with 4.0 Log(TIF)Click here for additional data file.S6 FigLocations of residues 125 (blue), 127 (red), 222 (yellow), 223 (green) and 380 (purple) were visualised using Pymol software (Protein Data Bank reference: 3UBE). Monomers are coloured in shades of grey and positions of HA substitutions have been highlighted in each monomer of the three-dimensional structure. Residues locations are shown from (A) a side view and (B) a top view of the HA trimer.(TIF)Click here for additional data file.S7 Fig2 titres were recorded.Additional accelerated thermostability assays were performed with A/SLOV15 N380D (dark blue) and A/SLOV15 S12 (light purple). A/SLOV15 V1 (light blue), A/SLOV15 S13 (dark purple), A/NC99 (black) and A/CA09 (red) LAIV viruses were included for reference. A/SLOV15 N380D, A/SLOV15 S12, A/SLOV15 V1, A/SLOV15 S13, A/NC99 and A/CA09 viruses were held at temperatures ranging from 47.5°C—65°C for 20 minutes. After incubation, all samples underwent haemagglutination assays using 0.5% chicken red blood cells and log(TIF)Click here for additional data file.


The conductive network constructed by CNFs not only facilitates electron transport, but also provides landing sites for nanoparticles, thus reducing nanoparticle aggregation and exposing more active sites. Additionally, the synergistic interaction between Mn3O4 and NiO improved electrocatalytic capacity for glucose oxidation. The Mn3O4/NiO/CNFs modified glassy carbon electrode shows satisfactory results in terms of linear range and anti-interference capability for glucose detection, suggesting that the constructed enzyme-free sensor has a promising application in clinical diagnosis.Transition metal oxides have garnered a lot of attention in the field of electrocatalysis along with their unique crystal structure and excellent catalytic properties. In this study, carbon nanofibers (CNFs) decorated with Mn Electrochemical glucose sensors perform an indispensable role in monitoring glucose concentrations, which not only can be employed to control the intake of glucose in diet, but also to predict diseases associated with high or low glucose levels ,2. Enzym2 utilization ,352/Ni(A3O4/NiO/CNFs. EIS tests of different modified electrodes were conducted in the solution with 5 mM [Fe(CN)6]3−/4− and 0.1 M KCl. The outcome of the EIS test is reflected by the Nyquist plot, where the semicircular part of the high frequency region relates to the electron transfer restriction process and the linear part of the low frequency region reflects the diffusion process. The decrease in the radius of the semicircle represents a decrease in the electron transfer resistance at the electrode interface (Ret). In 3O4/CNFs/GCE and NiO/CNFs/GCE, Mn3O4/NiO/CNFs/GCE shows the smallest diameter, indicating the fastest charge transfer capability. To investigate the electrochemical performance of Mn3O4/NiO/CNFs, CV was employed. 3O4/CNFs/GCE, NiO/CNFs/GCE, and Mn3O4/NiO/CNFs/GCE in 0.1 M NaOH solution with and without 2 mM glucose. The oxidation current that changed for Mn3O4/CNFs/GCE in the presence of glucose can be neglected. Meanwhile, NiO/CNFs/GCE exhibited a positive electrocatalytic oxidation peak current for glucose detection which can be ascribed to the transition between Ni2+ and Ni3+. Notably, Mn3O4/NiO/CNFs/GCE exhibited the highest oxidation current. The mechanism of glucose oxidation on Mn3O4/NiO/CNFs can be explained by the following equations [The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to study the electrochemical performance of Mnquations :NiO + H22 (Equation (1)). Next, Ni(OH)2 was oxidized to NiOOH as electron mediator (Equation (2)). Then, NiOOH obtained electrons from glucose to form Ni(OH)2, and glucose was oxidized to gluconolactone (Equation (3)). Previous studies have indicated that Mn-based materials have a wide range for enzyme-free detection of glucose [3O4 can increase the electrical conductivity of the composites.Originally, in the alkaline medium, NiO strongly adsorbed water molecules to form Ni(OH) glucose ,38. In t glucose . The res3O4/NiO/CNFs/GCE in different glucose concentrations (from 0 mM to 5 mM). The oxidation current signal increased with increasing glucose concentration, indicating that Mn3O4/NiO/CNFs are suitable as electrocatalytic media for the construction of glucose sensors. To further investigate the kinetic process of glucose oxidation on the electrode surface, the CV curves of Mn3O4/NiO/CNFs/GCE were tested in the range of scan rate from 20 mV s−1 to 320 mV s−1 (pa) and cathodic peak (Ipc) maintained a good linear connection with the square root of the scanning speed, indicating that the oxidation of glucose on Mn3O4/NiO/CNFs/GCE is a diffusion-controlled process.0 mV s−1 c. As ill3O4/NiO/CNFs on glucose detection, the time-current method was used to optimize the parameters during the experiments, including the applied voltage and the concentrations of NaOH and Mn3O4/NiO/CNFs. 3O4/NiO/CNFs is 9 mg mL−1; therefore, 9 mg mL−1 was selected as the optimum modification material concentration. 3O4/NiO/CNFs/GCE for continuous addition of glucose under known optimal experimental conditions. The relationship between response current and glucose concentration shows linearly in the range of 5–3000 μM (R2 = 0.997) and 3000–12,000 μM (R2 = 0.993), and could be well-fitted to the equation: I (μA) = 27.33 C (mM) + 1.343 and I (μA) = 17.22 C (mM) + 35.015. The detection limit of Mn3O4/NiO/CNFs/GCE was calculated to be 0.73 μM (S/N = 3). The sensitivity was calculated to be 386.84 μA mM−1 cm−2 (5–3000 μM) and 243.74 μA mM−1 cm−2 . Additionally, the response time is a key factor in weighing up the practical application of the sensor. 3O4/NiO/CNFs/GCE at different glucose concentrations reaches its steady-state current within 2–4 s after the addition of glucose. 3O4/NiO/CNFs/GCE has a wider linear range as well as lower detection limit as glucose sensor.To improve the electrocatalytic effect of Mn3O4/NiO/CNFs/GCE had good selectivity. 3O4/NiO/CNFs/GCE to 100 μM glucose. The current kept stable during 2000 s, suggesting that the electrode had an excellent stability as glucose sensor. Besides, repeatability and reproducibility are also key to determine whether the synthesized materials can be used for actual sample detection. The five identical modified electrodes used to detect 0.1 mM glucose were found to have a relative standard deviation (RSD) of 6.38% (3O4/NiO/CNFs/GCE was stored at room temperature for 30 days. The results show that the response current remains at 95.3% of the initial current after 30 days (The presence of other easily oxidized molecules in the actual sample may affect the accuracy of glucose detection. In of 6.38% , indicatof 6.38% , meaning 30 days , indicat3O4/NiO/CNFs/GCE, serum samples with different concentrations of glucose were detected using standard addition method. The serum samples used in the experiment were from the School of Medicine, Shanghai University. The recoveries of the three samples were 100.09%, 100.20%, and 99.91%, respectively (To evaluate the practicality of Mnectively , indicat3O4/NiO/CNFs were synthesized by electrospinning and following calcination, and were used as an enzyme-free sensor for glucose detection. The intricate network structure of CNFs provides the backbone for the nanoparticles and facilitates electron transport. In addition, the synergistic effect between Mn3O4 and NiO is beneficial to improve the catalytic performance for glucose detection. The constructed Mn3O4/NiO/CNFs/GCE sensor has good linear range, low detection limit, and anti-interference ability, indicating that Mn3O4/NiO/CNFs have some application prospects as electrochemical sensors in clinical diagnosis.In this work, Mn


CDKN2A/B deletions in childhood acute lymphoblastic leukemia (ALL), we retrospectively analyzed 819 consecutive B-ALL patients treated with the Chinese Children’s Cancer Group ALL-2015 (CCCG-ALL-2015) protocol, and fluorescence in situ hybridization (FISH) analysis on CDKN2A/B deletion was available for 599 patients. The prevalence of CDKN2A/B gene deletions was 20.2% (121/599) of B-ALL. CDKN2A/B deletions were significantly associated with older age, higher leukocyte counts, a higher percentage of hepatosplenomegaly, and a higher frequency of BCR-ABL (p < 0.05). Those patients achieved similar minimal residual disease (MRD) clearance and complete remission compared to patients without CDKN2A/B deletion. The CDKN2A/B deletions were correlated with inferior outcomes, including a 3-year event-free survival (EFS) rate  and a 3-year overall survival (OS) rate . In multivariable analysis, CDKN2A/B deletion was still an independent prognostic factor for EFS in total cohorts (p < 0.05). We also detected a multiplicative interaction between CDKN2A/B deletions and TP53 deletion on dismal prognosis (p-interaction < 0.05). In conclusion, CDKN2A/B deletion is associated with distinct characteristics and serves as a poor prognostic factor in pediatric ALL, especially in TP53 deletion carriers.To further emphasize the clinical–genetic features and prognosis of  CDKN2A/B is one of the most frequent abnormal genes in ALL and can be detected by fluorescence in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), array-based comparative genomic hybridization (aCGH) analysis, and single-nucleotide polymorphism array (SNPA) (CDKN2A/B deletions were found in approximately 20%–25% of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and 38.5%–50% of T-ALL patients  is one of the most curable malignancies, with 5-year event-free survival rates exceeding 80% in many developed countries and even exceeding 90% in high-income countries . With thy (SNPA) . As a sepatients , 8. Desptric ALL , 9, 10, eage ALL , 11. As The cohort included 902 patients with newly diagnosed pediatric ALL who were treated according to the CCCG-ALL 2015 protocol   register9/L, a chromosome number of more than 50, or the ETV6–RUNX1 fusion gene; and did not have CNS 3 status or testicular leukemia, and had a minimal residual disease of less than 1% on day 19 of induction and less than 0.01% on day 46 of induction. Patients with a minimal residual disease of 1% or more  in bone marrow on day 46 of induction and infants younger than 6 months with KMT2A rearrangement and a leukocyte count of 300 × 109/L or more were classified as high-risk ALL. The remaining participants were assigned to the intermediate-risk group  and contained many genes, including methylthioadenosine phosphorylase, CDKN2A (INK4A and ARF), and CDKN2B (INK4B) in the 9p21 chromosome region. The cutoff level for positive results was calculated to be 5%, and at least five hundred cells were analyzed , polymerase chain reaction (PCR) for fusion gene  , and karanalyzed . Some ca6 leukocytes from bone marrow were incubated with marker panel  for 30 min, washed twice, and then resuspended in 100 μl of PBS. The cells were acquired on a Navios  and analyzed using Kaluza software. Samples were defined as MRD-negative if no baseline and/or different-from-normal leukemia-associated immunophenotypes (LAIP) cells could be quantitated above the limit of detection (approximately 0.01%)  on days 19 and 46 during the first induction. A total of 1–2×10y 0.01%) .-5 was defined as positive. A poor response to prednisone was considered if the peripheral blast count on treatment day 5 was not lower than 1×109/L.Diagnosis of complete remission (CR) was based solely on bone marrow morphology with a cutoff value of 5% of leukemic blasts on treatment day 46. Bone marrow MRD that is not lower than 10The primary endpoint event-free survival was calculated from the date of diagnosis until the following events: induction treatment failure, any relapse after CR, second malignancy, and death due to any cause. Overall survival was considered as the time from diagnosis to death due to any cause.U tests were used to compare categorical and continuous variables, respectively. Event-free and overall survival were calculated and compared using Kaplan–Meier analysis and log-rank tests. Cox proportional hazard regression analyses were performed using univariable and multivariable regression approaches. Variables that were found statistically significant and a p-value of approximately 0.1 were included in multivariable Cox regression analysis. We performed subgroup analyses for prespecified baseline factors with rates of inferior events by factor interaction with the use of Cox models. Statistical significance was defined as a p-value of less than 0.05. All analyses were performed using SPSS v. 24. and SAS v. 9.4 software.Chi-squared test and Mann–Whitney CDKN2A/B gene deletions in B-ALL was 20.2% (121/599). Among patients with CDKN2A/B deletions, 57 (45.3%) harbored CDKN2A/B biallelic deletions, whereas 82 (54.7%) harbored monoallelic deletions, and 11 (7.3%) patients harbored both biallelic and monoallelic deletions.In total, 599 patients were included in the analysis. The final follow-up was in December 2020 and the median follow-up time was 34 months (range: 0 to 58 months). The median age was 5 years , and the male/female ratio was 1.4 (350/249). The prevalence of CDKN2A/B , a higher leukocyte count , a lower platelet count , and a higher percentage of hepatosplenomegaly . A higher rate of central nervous system status 2(CNS 2)/traumatic lumbar puncture was found among patients with CDKN2A/B deletion, but no statistical significance was found in the two groups. Patients with CDKN2A/B deletions belonged more to the intermediate-risk groups  than patients without CDKN2A/B deletions based on CCCG-ALL 2015 risk stratification. There was no significant difference between patients with biallelic deletions and monoallelic deletions. The comparison of characteristics for the two groups is shown in Compared to patients with wild-type CDKN2A/B Table 1,CDKN2A/B deletions with other cytogenetic abnormalities. The CDKN2A/B deletion group had a higher prevalence of the BCR/ABL fusion gene . A higher co-occurrence of the ETV6/RUNX1 fusion gene (19.8% vs. 26.8%) was found in patients with CDKN2A/B deletion; however, no statistical significance was identified. A higher incidence of chromosome < 44 (2.5% vs. 0.6%) and TP53 deletions (8.3% vs. 4.0%) was detected by FISH, but this did not reach statistical significance (p ≥ 0.05). The genetic feature of patients with CDKN2A/B gene deletions was also related to the risk stratification distribution at the first diagnosis.Karyotype analysis, 43 fusion genes, and FISH studies of pretreatment bone marrow were available for 599 patients. p = 0.091) in the CDKN2A/B deletion group. No significant difference was observed between the two groups for MRD on day 19  and 46 . The CR rate  for each group was equivalent.A chi-squared test showed a high rate of poor responses to prednisone (PPR) . Six patients died [n = 2 died of severe pneumonia and emesis in remission and n = 4 died after chimeric antigen receptor T-cell immunotherapy (CAR-T) therapy or hematopoietic stem cell transplantation (HSCT) because of elevated MRD level]. The 3-year OS and EFS were 93.6 ± 1.1% and 85.2 ± 1.6%, respectively, for the whole series.A high CR rate was achieved in 593 of 599 patients (99.0%) Figure 1CDKN2A/B deletion group is related to a higher relapse rate  . Patients with CDKN2A/B deletions had lower 3-year EFS and OS rates than patients without CDKN2A/B deletions   for EFS in whole cohorts (p = 0.000) for all cases.The = 0.000) Table 2.CDKN2A/B deletion group at diagnosis. For patients assigned into the low-risk group, patients with CDKN2A/B deletions had adverse clinical outcomes  compared to patients without CDKN2A/B deletions , but no inferior 3-year OS  compared to intermediate-risk patients without CDKN2A/B deletions , while there were no significant interactions between two groups (p-interaction = 0.0631).We performed stratified analyses by subgroups defined by major co-variables that might have been related to EFS and further quantified the effect modification of major co-variables on the influence of variates Table 3.CDKN2A/B deletions treated in a single center and demonstrated the adverse effect of CDKN2A/B deletions on clinical outcomes.In this study, we present a large retrospective study of pediatric ALL patients with CDKN2A/B deletions in our study was 20.2%, which was higher than Agarwal’s research (19.8%) but lower than Sulong’s research (22.0%)  . Previou2.0% . Proversial , 10, 19.CDKN2A/B deletions in childhood ALL were associated with an increased probability of relapse and death  than patients without biallelic deletions  compared to low-risk patients without CDKN2A/B deletions  has improved overall survival than chemotherapy alone did (n = 20) in relapse patients with CDKN2A/B deletions, though no significance was observed between the two groups   in ph+ patients for the entire duration of ALL therapy  ALL  ALL , 25; furCDKN2A/B deletions were more frequently steroid-resistant, whereas they had a better MRD clearance on day 19 of induction therapy compared with patients without CDKN2A/B deletions. These results differed from a study by Braun who found higher MRD levels on day 15 of induction therapy in patients with CDKN2A/B deletions  combined with chemotherapy are in clinical trials for the management of pediatric patients with relapsed/refractory ALL , 81870131 (XZ), and 81670112 (XZ).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Yeast expressing non-phosphorylatable Spc110S11A had a distinct spindle phenotype characterised by higher levels of α-tubulin, which was frequently asymmetrically distributed between the two SPBs. Furthermore, expression of the double mutant Spc110S11AS36A had a delayed cell cycle progression. Specifically, the final steps of mitosis were delayed in Spc110S11AS36A cells, including expression and degradation of the mitotic cyclin Clb2, disassembling the mitotic spindle and re-localizing Cdc14 to the nucleoli, resulting in late mitotic exit and entry in G1. Thus, we propose that Spc110 phosphorylation at S11 and S36 is required to regulate timely cell cycle progression in budding yeast.Spc110 is an essential component of the spindle pole body (SPB), the yeast equivalent of the centrosome, that recruits the γ-tubulin complex to the nuclear side of the SPB to produce the microtubules that form the mitotic spindle. Here, we identified phosphosites S11 and S36 in maternally originated Spc110 and explored their functions This article has an associated First Person interview with the first author of the paper. Summary: We identified two phosphosites (S11 and S36) in the yeast centrosome component Spc110 from maternal origin and found that these phosphosites control timely cell cycle progression. Centrosomes and yeast spindle pole bodies (SPBs) are known for their conserved roles in microtubule (MT) nucleation and spindle formation for chromosome segregation. In budding yeast, the SPB is a layered structure inserted in the nuclear envelope throughout mitosis . The MTsMoreover, centrosomes and SPBs serve as signalling platforms for cell cycle regulators, checkpoint proteins and MT-associated proteins, and have a direct role in cell cycle regulation . For exaPrevious research has shown that all the SPB components are phosphoproteins. In total, 297 phosphosites have been identified by phosphoproteomic analysis from intact SPBs, and many of these sites are cell cycle specific . HoweverThe phosphorylation of Tub4, Spc72 and Spc110 that regulate MT nucleation are some of the best functionally characterised phosphosites of the SPB . Spc110 Experiments using fluorescence recovery after photobleaching (FRAP) have shown that Spc110 undergoes a dynamic exchange in and out of the SPB in G1/S until the spindle is fully assembled, when Spc110 becomes stable . In a prHere, we applied RITE to purify stable, old  Spc110 for mass spectrometry analysis. Our studies identify two phosphosites in old Spc110 that regulate cell cycle progression for timely mitotic exit.SPC110 gene  Spc110, we generated a yeast RITE strain expressing epitope tags amenable for affinity-based purification. We used a yeast strain that constitutively expresses Cre-recombinase fused to the estrogen-binding domain (EBD), Cre-EBD78 previously described , and int110 gene A. The RITo coordinate the epitope tag exchange with the SPB duplication, we used the RITE assay previously developed to follow SPB inheritance  Fig. 1BBFig. 1B.We next set up a protocol to purify Spc110-Flag and Spc110-V5 D. We col2) beads. The MT nucleating Spc110 phosphorylation sites, the Mps1 (S60 T64 T68), and Cdk1 (S36 and S91) sites lie in the Spc110 N-terminal region  and Spc110-V5 (newly synthesised) in three biological replicates, followed by mass spectrometry analyses. First, we enriched the purified samples for phospho-peptides using titanium dioxide  samples, we focused our analyses on Spc110-Flag . We attained a sequence coverage of 45-50%  in the tSPC110 is essential for growth, these results indicate that the mutants do not affect the protein's expression and folding.Spc110 phosphosite pS11 has been recently reported in a high-throughput study using DNA damage conditions , but itsSPC110 mutant alleles in spindle formation and cell cycle progression, we imaged cells expressing Spc110-superfolderGFP (sfGFP) [wild-type (WT) and mutant alleles] and mRuby2-Tub1 . We synchronised the cells in G1 with α-factor, released them in fresh media, and fixed them at 10-min intervals for microscopy. Spc110-sfGFP had the same localisation and intensity in the mutants as the WT strain  plots were similar for WT and mutant cells, indicating that all the cells exit the G1 arrest with similar kinetics (S11AS36A-sfGFP cells and had a higher number of cells in anaphase than WT cells at 70 min (29% versus 15%) and 80 min (18% versus 8%).We then scored the number of cells in each cell cycle phase according to the spindle length (mRuby2-Tub1). Progression through metaphase was similar for all cells, with a sharp peak at 40 min post-release  or Spc110anaphase A-C. We v one SPB B.S11AS36A cells followed the same pattern but were slightly delayed, starting with 38% Type 0 cells and reaching 60% cells in G1.During the release, we observed different patterns of Cdc14 localisation A. In latS11AS36A cells have a slower cell cycle progression resulting in a delay entering the next G1  and a higher percentage of Type 3 cells (30%).Lastly, we repeated the cell cycle and Cdc14 analyses in α-factor synchronised cells by adding α-factor during the release once the cells had budded (40 min post-release), thereby arresting the cells in the next G1 . This ex next G1 , in agre next G1 . The deltic exit . SpecifiIn this work, we applied the RITE method to separate old (Flag-tagged) and new (V5-tagged) Spc110 with four steps: (1) RITE-assay, (2) TAP-enrichment of intact SPBs, (3) denaturing SPB complex and (4) Flag- and V5-immunoprecipitation. Further mass spectrometry analyses identified three phosphosites  in Spc110-Flag. Therefore, considering the flow cytometry analysis of our samples C, we intThe S11 phosphosite of Spc110 was absent in previous comprehensive phospho-proteomic analyses of the SPB . One posS36A and Spc110S11AS36A had a delayed SPB separation  occurs from S-phase and stimulates γ-TuSC nucleation and spindle assembly . Followi old SPB B-C. OverS11AS36A was a delay in late anaphase and telophase  and the protein kinase Cdc15, the effector of the MEN pathway, was predicted as a potential kinase for Spc110-S11. Therefore, we hypothesise that phosphorylation of Spc110 at S11 is a target of the MEN pathway. This would explain why we identify this phosphosite in old (Flag-tagged) Spc110, primarily located at the old SPB where MEN is activated. However, during MEN activation, Cdc15 is docked to the cytoplasmic side of the SPB. It remains to be investigated whether there is an active pool of Cdc15 in the nucleus.Interestingly, we searched for kinase consensus sites in Spc110 using Scansite 4  were tagged by PCR-mediated homologous recombination and confirmed by sequencing. The mRuby2-Tub1 and mTurquoise2-Tub1 strains were constructed by integrating the plasmids pHIS3p-mRuby2-Tub1 (Addgene #50645) and pHIS3-pmTurquoise-Tub1 (Addgene #50641) at the TUB1 locus, as previously described  twice, with a 60 min interval. Cells were released by washing with YEP, resuspending in YEP, and incubating at 30°C. To rearrest the cells in the next G1, 3 µg/ml α-factor was added again to the cultures 40 min after the release.Cells were grown at 30°C in YEP  medium supplemented with 2% glucose. For synchronisation, overnight cultures were diluted to ODCells (1 ml) were fixed in 70% ethanol on ice for 1 h, resuspended in 50 mM sodium citrate (pH 7) for 15 min at RT, and incubated with 10 mg/ml RNase A  overnight at 37°C. The cells were washed with PBS, stained with 50 µg/ml propidium iodide  in 100 µl PBS, and sonicated for five pulses . DNA content was analysed using a flow cytometer  and FlowJo software.The RITE-genetic switch was induced as described before . BrieflyTM SamplePrep Freezer/Mill, using 10 rounds of 2 min grinding at 14 counts per second (cps) and 2 min cooling. Spc97-TAP purification of SPBs was performed as previously described (24) and the SPBs were eluted with 5 μg proTEV plus protease  in TEV buffer overnight at 4°C. Trichloroacetic acid (TCA) was added to a 10% concentration, proteins were precipitated on ice for 1 h and washed with ice-cold acetone. The pellet was air-dried, dissolved in B0 buffer , and boiled at 95°C for 10 min.For protein purification, the cells were pelleted, resuspended in 800 μl TAP purification buffer  and lysed in a SPEXFor affinity purification, the samples were diluted in IP buffer , and incubated with Protein G Dynabeads (Thermo Fisher Scientific) conjugated with Flag M2 mouse monoclonal antibody  or V5 tag mouse monoclonal antibody  at RT for 30 min. The beads were washed with wash buffer  four times.Proteins were separated by SDS-PAGE, transferred to a PVDF membrane (BioRad) and immunoblotted with primary monoclonal anti-FLAG M2 antibody ; V5 tag mouse monoclonal antibody , Pgk1 monoclonal mouse antibody  and HRP-conjugated secondary antibodies, detected with Femto maximum sensitivity substrate (Thermo Fisher Scientific). ImageLab software (Bio-Rad) was used for image acquisition and densitometric analysis. The density of a given band was measured as the total volume under the peak and corrected with background subtraction. The intensity was normalised with respect to corresponding Pgk1 signals of each sample.Samples were denatured and reduced with 50% trifluoroethanol (TFE) and 20 mM Tris (2-carboxyethyl) phosphine (TCEP) in 50 mM triethylammonium bicarbonate (TEAB) for 20 min at 55°C, then alkylated by 55 mM chloroacetamide (CAA) at RT for 30 min in the dark, followed by digestion with 5 mg of Lys C for 4 h and 5 mg modified trypsin overnight at 37°C. After digestion, the samples were centrifuged at 20,000 g for 10 mins to collect the peptide supernatant. The peptides were acidified with 10% trifluoroacetic acid (TFA) to pH 2, then desalted with a C18 StageTip . The stage tip was activated with 100% acetonitrile and equilibrated with 0.5% acetic acid. The peptide sample was then loaded onto the column and washed with 0.5% acetic acid. The bound peptides were eluted with 65% acetonitrile in 0.5% acetic acid solution into a fresh 1.5 ml Eppendorf tube.2 beads. The samples for phospho-enrichment were resuspended to the final composition of 6% TFA in 40% acetonitrile and incubated with the beads at room temperature (RT) for 10 mins. This incubation was repeated once with fresh beads. The beads were loaded over a C8 mesh, washed with 6% TFA in 10%, 40% and 60% acetonitrile sequentially, and eluted twice with 5% ammonia water first, followed by 5% ammonia water in 25% acetonitrile. The phosphopeptides were acidified and filtered to remove the TiO2 beads with a C8 frit prior to sample acquisition.Phosphopeptides were enriched from the desalted peptides with TiONon-enriched total peptides were analysed by mass spectrometry analysis using reverse-phase liquid chromatography on a Proxeon 1000 UHPLC system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Each fraction was separated on 50 cm×75 µm Easy Spray column (Thermo Fisher Scientific) in a 70-min gradient with a pre-programmed mixing of solvent A (0.1% formic acid in water) and solvent B . The following acquisition parameters were applied: data-dependent acquisition in positive mode with survey scan of 120,000, scan range of 350-1550 m/z, and AGC target of 4e5; MS/MS collision-induced dissociation in an ion trap and AGC target of 5e4; isolation window 1 m/z.All phosphopeptides samples were subjected to mass spectrometry analysis using reverse-phase liquid chromatography on an nLC 1000 UHPLC system connected to Q Exactive HF-X Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Separation column and solvent gradient setting are the same as above-mentioned on Lumos mass spectrometry. The only different acquisition parameters were: AGC target of 5e5 for full MS scan; and 32% HCD energy used for precursor fragmentation.Saccharomyces cerevisiae database (7939 entries). Search parameters: MS precursor mass tolerance 10 ppm, MS/MS 0.06 Da, maximal three missed cleavages; static modifications: carbamidomethyl (C); variable modifications: oxidation (M), deamidated (NQ), phospho (ST), phospho (Y) and acetyl N-terminal protein. False discovery rate estimation with two levels: strict=1% FDR and medium=5% FDR.Peak lists were generated in Proteome Discoverer 2.3 software (Thermo Fisher Scientific) with Mascot 2.6.0 (Matrix Science) and concatenated forward/decoy Cells were fixed in 4% formaldehyde at RT for 15 min, washed with PBS, and stored in PBS at 4°C. Fixed cells were then plated in concanavalin-A-coated 96-well glass-bottomed plates (Mo Bi Tec 5242-20) and imaged in PBS. Z-stacks  were acquired using a Nikon Ti2 inverted microscope equipped with a Kinetix sCMOS camera and a 60× water immersion objective (1.20 NA).Maximum intensity projections of image z-stacks were made with FiJi. Cell cycle analyses were performed by classifying the cells: G0/G1 (unbudded); G1/S ; metaphase ; early anaphase ; late anaphase ; and telophase . The fluorescence intensities of Spc42-tagRFP-T and Spc110-sfGFP were quantified with FiJi, segmenting SPBs by Spc42-tagRFP-T intensity. The regions of interest (ROIs) were used to measure integrated fluorescence intensities in the red (Spc42-tagRFPT-T) and green (Spc110-sfGFP) channels. Background correction was calculated by multiplying the mean fluorescence in the cytosol with the ROI's area and subtracting it from the integrated intensity. Cdc14-tagRFP-T, Spc110-sfGFP and mRuby2-Tub1 fluorescence intensities were quantified using the same approach, segmenting areas of G1 SPBs  or G1 nucleoli . Early anaphase spindles were analysed by first applying a directional filter and then segmenting using the mRuby2-Tub1 signal intensity.10.1242/biolopen.059565_sup1Supplementary informationClick here for additional data file.


Targeting these receptors preclinically can mitigate pathogenic β-amyloid and tau neurotoxicity whilst improving cognition and memory. In this review, we provide an accessible summary of the literature on Alzheimer’s disease and the therapeutic potential of A1 and A2A receptors. Although there are no available medicines targeting these receptors approved for treating dementia, we provide insights into some novel strategies, including allosterism and the targeting of oligomers, which may increase drug discovery success and enhance the therapeutic response.Alzheimer’s disease (AD) is the most common dementia in the elderly and its increasing prevalence presents treatment challenges. Despite a better understanding of the disease, the current mainstay of treatment cannot modify pathogenesis or effectively address the associated cognitive and memory deficits. Emerging evidence suggests adenosine G protein-coupled receptors (GPCRs) are promising therapeutic targets for Alzheimer’s disease. The adenosine A Dementias are the most prevalent neurological disorder in the aged population, affecting more than 50 million people worldwide in 2020 . In the Compared to age-matched non-AD individuals, AD patients experience an accelerated rate of brain mass shrinkage due to neuronal death and grey matter loss; the amount of neuronal atrophy correlates with AD severity –11. AD nThe amyloid cascade hypothesis posits the accumulation of toxic amyloid β (Aβ) peptides as the main cause of neurodegeneration . The forAn opposing view suggests tau pathology is responsible for the initiation of AD neurodegeneration. Under normal conditions, soluble tau protein acts as a microtubule stabiliser, maintaining neuronal integrity, neurite outgrowth, and axonal transport –27. The Another pathological feature shared by all AD pathogenesis theories is chronic inflammation. The inflammation theory of AD pathogenesis postulates that activated microglia and reactive astrocytes trigger AD pathogenesis and this event precedes the presence of Aβ plaques and NFTs . This th2+) entry and upregulation of pro-inflammatory mediators . P. P1R and regions –149. Thefrom ATP . Conversysiology , 152.Fig1R and A2AR also signal via additional effectors, including mitogen-activated protein kinases (MAPK) and Akt/protein kinase B, which play a role in cell growth, survival, differentiation, and protein transcription and translation [1R stimulates the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinases (JNK), and the p38 MAPK, and additionally phosphorylates Akt via phosphoinositide 3-kinase (PI3K) activation [i/o proteins. Additionally, A1R mobilises intracellular calcium stores and activates multiple isoforms of protein kinase C, via direct βγ interactions with phospholipase C [2AR also signals via ERK1/2, JNK, p38, and Akt, with these effects thought to be downstream of α subunit activity of Gs [2AR, suggesting this signalling can be conditional, depending on cell background and disease context [In addition to cAMP signalling, both Anslation , 154. A1tivation –162. Thilipase C . Similarty of Gs , 166–173 context , 175.1R and A2AR both stimulate additional pathways and effectors that are either brain-specific or present in only niche cell types. A2AR has been established as a modulator of neurotrophins, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which play important roles in neuronal differentiation and survival, in addition to regulating synaptic transmission and plasticity [2AR can increase the release of both BDNF and NGF, which may be mediated by the transactivation of neurotrophic receptors [1R has also been suggested to play a role in neurotrophin signalling, although its effects are less well defined [In addition to the above-generalised signalling pathways, Aasticity . In micreceptors –181. A1R defined .1R and A2AR. A1R couples to a number of ion channels mediating an overall net hyperpolarisation of neurons [1R couples to G protein-coupled inwardly-rectifying potassium (GIRK) channels, which are a class of transmembrane proteins that facilitate potassium flux into the cytosol [1R coupling to GIRK channels is through the direct binding of βγ subunits. Additionally, A1R reduces the current of voltage-dependent calcium channels, with a proposed preference for N-type channels [1R, A2AR also couples to voltage-dependent calcium channels; however, A2AR has a stimulatory effect on the current [ATP), which are present in plasma, mitochondrial, and nuclear membranes, and play a role in neuronal excitability and survival [Modulation of ion channel activity represents another vital signalling mechanism for both A neurons . It is w cytosol –186. A1Rchannels –190. Alo current , 191. Bosurvival , 193.1Rs and excitatory effects of A2ARs, allowing fine-tuning of neuronal circuitry. Activation of presynaptic A1Rs on excitatory neurons reduces neurotransmitter release and induces synaptic depression, whereas presynaptic A2ARs are involved in increasing neurotransmitter release [1R activation causes membrane hyperpolarisation with subsequent inhibition of neuronal firing. In contrast, activated postsynaptic A2ARs increase cellular excitability [1R and A2AR are not limited to the modulation of neuronal activity, these receptors also coordinate the function of additional cells, including astrocytes and microglia . In animal models, including APP/PS1 mice and THY-Tau22 mice, administering selective A2AR antagonists improved memory deficits and mitigated the Aβ toxicity or tau hyperphosphorylation associated with the disease [1-42 fragment reduced memory performance in rats, which was reversed by SCH58261 and KW6002 [2AR antagonism could not mitigate acute memory deficits induced by the muscarinic receptor antagonist scopolamine or the NMDA antagonist MK801. As such, it has been suggested the beneficial effects of A2AR antagonists on memory may not be a generalised effect but rather specific to the disease processes involved in dementia/AD.Epidemiological studies have reported an inverse association between caffeine intake and AD/dementia risk –217. Sigectively , 219. Inectively , 212. Lon levels . These n disease , 221. Mod KW6002 . Interes2AR blockage in neurons attenuated Aβ-induced neuronal death and further reduced synaptic loss [1R antagonist CPX was ineffective, implying Aβ-induced neurotoxicity is primarily mediated through A2AR activity [1R. In a model using human neural cells, application of the selective A1R agonist R-PIA led to soluble APP production and increased tau phosphorylation, which was reversed by A1R-selective antagonist DPCPX [1R antagonist rolofylline restored memory deficits and reduced synaptic dysfunction in neural cells [1R redistributes and co-localises with NFTs and Aβ plagues in AD patients, these studies suggest A1R may play a direct role in mediating some of the pathology [1R activation is strongly neuroprotective in a number of settings [1R agonist decreased neurodegeneration in in vitro and in vivo models challenged with noxious stimuli [1R antagonists may protect against β-amyloid and tau neurotoxicity and enhance cognition, whereas A1R agonists may impart neuroprotection.Alongside animal studies, pharmacological Atic loss . Similaractivity . Additiost DPCPX . In tau al cells . Given tathology , 202. Imsettings  and elicsettings , 227. In2AR genetic deletion or overexpression has also revealed interesting findings. In APP/PS1 mice, A2AR downregulation via shRNA restored long-term potentiation and improved memory deficits [2AR overexpression in cortical and hippocampal neurons of rats resulted in increased glutamate release, which was associated with changes in synaptic plasticity [2AR protected against spatial memory deficits, reduced neuroinflammation, and decreased tau hyperphosphorylation [2AR overexpression in the forebrain of THY-Tau22 mice resulted in tau hyperphosphorylation and increased memory deficits [2AR may contribute to this process. Interestingly, upregulation of genes associated with immune responses was also found in this study, with further cell-specific enrichment analysis revealing these genes were preferentially increased in microglia. As such, microglial A2AR and its associated immune responses may play a role in the pathogenesis of dementia. Indeed, additional studies support the importance of glial cells, with A2AR being upregulated in microglia and astrocytes after treatment with Aβo [2AR enhancing long-term memory in young and in ageing mice [2AR in microglia offers an upstream therapeutic target [2AR activation stimulates sustained NLRP3 inflammasome activity and the production of proinflammatory cytokines (e.g. IL-1β) in macrophages and primary microglia [2AR on microglia may present an opportunity to target neuroinflammation associated with AD.In addition to pharmacological studies, Adeficits . In contasticity . In THY-rylation . Conversdeficits . Mice alwith Aβo  and conding mice . Targetiing mice , with emc target . A2AR acicroglia –235. Noticroglia , 237. Th1R and A2AR in dementia/AD highlights these GPCRs represent promising drug targets. Overall, the studies described predominantly indicate that A1R and A2AR antagonism may be a viable therapeutic approach to mitigate pathology and improve patient symptoms. Indeed, A1R antagonism may afford protection against β-amyloid and tau neurotoxicity and the enhancement of cognition is clearly desirable in the setting of dementia. However, the neuroprotective nature of A1R is also an important consideration. Studies showing reduced brain A1R expression in patients with AD make this particularly pertinent, as reduced A1R expression could facilitate neuronal excitotoxicity [2AR antagonism against β-amyloid neurotoxicity and memory impairment in preclinical settings makes it a desirable drug target. The A2AR antagonist istradefylline enhanced cognition in mice with amyloid pathology and is a clinically used adjunctive therapy in Parkinson’s disease [2AR agonism may be useful very early in the disease process, where its anti-inflammatory effects may dampen disease progression [2AR induction of BDNF signalling could also contribute to greater neuronal survival and maintenance of synaptic plasticity; however, these considerations remain largely unexplored.The growing evidence supporting a role for Atoxicity . As such disease , 240. Isgression . A2AR in1R or A2AR for AD/dementias remains apparent. This may be partly owing to the need for a deeper understanding of the precise roles of A1R and A2AR in AD/dementia. Moreover, the widespread distribution of ARs and their role in fundamental biological processes is an additional hurdle for the drug discovery process. For example, A1R activation is associated with lowered heart rate and altered blood pressure [2AR antagonism, such as that mediated by istradefylline, can cause involuntary movements and hallucinations [Nonetheless, despite the promising preclinical data, the void of clinically approved drugs targeting Apressure , whilst pressure , 244. A2inations . Many ofAllosteric ligands may overcome many of the limitations associated with traditional agonists and antagonists. Allosteric compounds bind to a topographically distinct site acting to modulate the affinity and/or efficacy of the orthosteric endogenous ligand, in this case, adenosine . A largeAllosteric ligands offer numerous advantages. Since allosteric sites typically show greater sequence divergence compared to orthosteric sites, allosteric ligands typically exhibit greater subtype selectivity , 248. As1R and A2AR, although there has been significantly more traction at A1R. A large number of A1R PAMs have been discovered, with very early studies characterising the allosteric enhancer PD 81,723 [1R PAMs has been demonstrated preclinically and in a phase II trial, confirming this pharmacological approach has the translational potential [1R NAMs have not been identified. A2AR allosteric modulators are limited. A fragment-based drug discovery approach identified potential PAM and NAM scaffolds for A2AR [1R and A2AR allosteric ligands. Despite the paucity of A1R and A2AR NAMs and the lack of evaluation of these classes of compounds in AD models, the physiological advantages remain conceptually promising. For A1R and A2AR NAMs, inhibiting signalling only where there is increased adenosine tone could mitigate β-amyloid neurotoxicity and enhance cognition, whilst avoiding the risk of seizures, sleep, and motor disturbances commonly associated with antagonism. This could afford a much more nuanced fine-tuning of AR signalling, without globally reducing adenosine function across all brain areas. Furthermore, this would also likely reduce the incidence of peripheral cardiovascular side effects.Allostery has been detected and quantified at both AD 81,723 . The utiotential , 251. Tofor A2AR . These ffor A2AR –257, are1R and A2AR is protein–protein interactions. The traditional model of GPCR activity depicted the receptors to function exclusively as monomeric entities. Over the last two decades, increasing evidence indicates GPCRs form homomers and heteromers or higher-order oligomers as part of GPCR normal trafficking and function [1R homomers at the plasma membrane using bimolecular fluorescence complementation and fluorescence correlation spectroscopy [2AR homomers were confirmed in bioluminescence resonance energy transfer (BRET) or Förster resonance energy transfer (FRET) experiments [2AR protomers [Another form of allostery to consider when targeting Afunction , 259. Hotroscopy . Converseriments  and may rotomers . Heteromrotomers ), as welrotomers .2AR agonists decrease the affinity and intrinsic efficacy of D2R agonists in A2AR-D2R heteromers [2R selectively confers negative cooperativity towards the A2AR antagonist SCH442416 in a A2AR-D2R heteromer, compared to when not forming heteromers or forming heteromers with A1R [2AR-D2R heteromers expressed in striatopallidal neurons, compared to presynaptic A2AR-A1R heteromers localised in cortico-striatal glutamatergic neurons. Heteromers/homomers with allosteric properties provide an exciting possibility to fine-tune receptor signalling, trafficking, and pharmacological properties.There are two general mechanisms by which receptor oligomerisation can influence the drug effect . (1) Theteromers . (2) Onewith A1R . As such2AR-D2R heteromers is a well-established therapeutic strategy for Parkinson’s disease where A2AR antagonism in conjunction with D2R agonism is highly desired. Commonly, A2AR antagonists are dosed in conjunction with L-DOPA; however, there are continuing efforts to develop bivalent ligands to co-occupy and specifically target A2AR-D2R heteromers [2AR-D2R heteromers, multiple bona fide AR heteromeric complexes are of therapeutic interest and relevant in the setting of AD  are increased in activated microglia as well as in the hippocampus of transgenic AD mice  [2AR has a central role in modulating mGlu5, D1R, and NMDAR signalling in the hippocampus under physiological conditions [2AR to form heteromeric complexes.As discussed above, A synapse , 274. A1icroglia . To therischemia , 277. Tasw, Ind) . A2AR handitions , possibl1R and A2AR as promising GPCR targets for Alzheimer’s disease. Overall, A1R and A2AR inhibition seems to mitigate the neurotoxicity associated with the β-amyloid accumulation and tau hyperphosphorylation and improve cognition and memory. Stimulation of A1R can also promote neuroprotection and therefore adds an additional layer of complexity in considering agonism versus antagonism when targeting this receptor. Although not explored for AD, the notion of biased agonism, which is a growing paradigm at adenosine receptors [1R compound to improve cognition but also remain neuroprotective. Indeed, although drug discovery at adenosine receptors has traditionally experienced hurdles, the novel therapeutic paradigms covered in this review, including allostery and the targeting of oligomers, present a promising future avenue of investigation. It is hoped harnessing this knowledge may increase the development and translation of clinical candidates with enhanced therapeutic responses and limited side effects.Dementia remains a significant neurological disorder. This review has highlighted preclinical studies implicating Aeceptors , 280, co


Penicillium oxalicum has been reported as a multienzyme-producing fungus and is widely used in industry due to great potential for cellulase release. Until now, there are only 10 available genome assemblies of P. oxalicum species deposited in the GenBank database. In this study, the genome of the I1R1 strain isolated from the root of Ixora chinensis was completely sequenced by Pacbio Sequel sequencing technology, assembled into 8 chromosomes with the genome size of 30.8 Mb, as well as a mitogenome of 26 kb. The structural and functional analyses of the I1R1 genome revealed gene model annotations encoding an enzyme set involved in significant metabolic processes, along with cytochrome P450s and secondary metabolite biosynthesis. The comparative analysis of the P. oxalicum species based on orthology and gene family duplications indicated their large and closed pan-genome of 9,500 orthologous groups. This is valuable data for future phylogenetic and population genomics studies. Penicillium, a genus of ascomycetous fungi, first described by Penicillium genus, many are involved in the decomposition saprophytes of organic materials. Several reports show that some Penicillium spp. can display negative impacts to the food crop as pathogens  is an anamorphic species of the genus Penicillium, which was named based on the feature of oxalic acid production , at temperature of −80°C, at the National Key Laboratory of Gene Technology . The P. oxalicum I1R1 assembly was evaluated in comparison with other published fungal genomic sequences belonging to the P. oxalicum species. Annotation was also examined to provide valuable information for efforts to determine the potential functions of P. oxalicum.In this study, we present the de novo sequencing of the whole genome of Penicillium sp. I1R1 strain isolated from the root of I. chinensis was cultured on Potato dextrose agar (PDA) media at 28°C ± 2 from glycerol stock and used for DNA extraction after 14 days of cultivation. I1R1 high-molecular-weight genomic DNA was collected by using Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit according to the manufacturer's instructions. A total 100 mg sample was used for each extraction. Before being used as a template for DNA library preparation, DNA product was quantified via 0.8% agarose gel electrophoresis using Pippin pulse—SAGE, Qubit dsDNA HS Assay Kits by QUBIT 3.0, and taxonomical reconfirmed through amplification of ITS1-5.8S-2 region and Sanger sequencing.The A total of 500 ng of I1R1 genomic DNA diluted in 150 µl Elution Buffer buffer was sheared by the g-TUBE (Covaris) into small fragments of 10 kb, and checked via Bioanalyzer Agilent 2100 with Agilent DNA 12000 Kit (5067-1508 Agilent). The sequencing library was prepared by SMRTbell Express Template Prep Kit 2.0 , followed by polymerase attached and purification by the Sequel Binding and Internal Ctrl Kit 3.0 . Calculation and setting up for loading on Sample Plate  with genomic DNA (concentration of 9 pM) was performed on Sample Setup software in SMRTLink portal version 9.0. The fungal genome was sequenced by PacBio SEQUEL with the Sequel SMRT Cell 1 M v3 Tray chip  and Sequel Sequencing Kit 3.0  at the Institution of Biotechnology, Vietnam Academy of Science and Technology .https://github.com/JanaSperschneider/FindTelomeres). Graphic representation of the I1R1 assembled chromosomes was visualized via the software Geneious Prime version 2021.2.2 (https://www.geneious.com/). Whole-genome alignment between P. oxalicum I1R1 and the reference strain CCTCC M 20211203 (GenBank assembly accession: GCA_021133555.1) was conducted and visualized by MAUVE . Long read mapping to the reference Penicillium citrinum mitochondria (NCBI Reference Sequence: NC_047444.1) was accomplished using pbmm2 (Penicillium chrysogenum reference genome ASM71027v1 (GenBank: GCA_000710275.1) to automatically generate full gene structure annotations in novel genomes. The sequences of ribosomal RNAs (rRNAs) were identified by barrnap prediction software version 0.9 through the whole-genome sequence  DNA sequencing were assembled using the hierarchical genome-assembly process version 4 for high-quality de novo fungal genome assembly . The mitochondrial annotation was executed via Mitos webtool version 2 , Aspergillus oryzae (NC_008282.1), Aspergillus fumigatus (NC_017016.1), Aspergillus niger (NC_007445.1), Aspergillus tubingensi (NC_007597.1), Aspergillus kawachii IFO 4308 (AP012272.1), Aspergillus nidulans FGSC A4 (JQ435097.1), Penicillium sp. ShG4C (KX931017.1), P. citrinum (MK919205.1), Penicillium polonicum (KU530219.1), Penicillium digitatum strain pd01 (HQ622809.1), and Cladosporium sphaerospermum (NC_050879.1). Multiple alignment of those sequences was performed using the MAFFT online tool .We sequenced the ITS1-5.8S-ITS2 region of the I1R1 strain by the primer ITS4/ITS5 for standard fungal taxonomy. We extracted ITS sequences by ITSx  deposited to the database. The genome sizes vary from 29.83 to 31.25 Mb. Different sequencing technologies greatly affect the number of generated contigs after assembly  shows that the genome assembly of I1R1 has the gene space completeness of 95% which represents high-quality assembly, similar to the data of 94.9% of the representative species P. oxalicum HP7-1. The new I1R1 assembly was equal in size to the HP7-1 assembly with 4.88% of the total-genome sequence being the repetitive regions . A high bootstrap value of 100 supports all branches of this clade  assigned into 6 levels, including 45 KEGG pathways  superfamily consists of enzymes involved in a variety of physiological processes, including xenobiotic degradation, detoxification, and biosynthesis of secondary metabolites  among the P. oxalicum strains, consisting of 106 specific gene models belonging to the P. oxalicum I1R1 genome. Orthologous analysis is the most accurate approach to investigate the similarities and differences between model organisms in order to infer the functional genetic information and propose function in novel sequenced genomes , 72 auxiliary activities (AAs), 44 carbohydrate-binding modules (CBMs), 44 carbohydrate esterases (CEs), 170 glycosyl transferases (GTs), and 11 polysaccharide lyases , cellulose (107), and pectin , due to the presence of CAZymes in GH79, GH88 families , naphthopyrone (100%), an antifungal compound AbT1 (100%), and nidulanin A (75%). Yellow pigments (naphthopyrones) are commonly found in Aspergillus sp., Fusarium sp., and several plants (P. oxalicum species can be engineered to produce oxaleimide (Genome mining of secondary metabolites was performed using antiSMASH. There were 36 found regions inside the aleimide .P. oxalicum I1R1 genome. We present the whole-genome sequence and annotation of the I1R1 comprising eight chromosomes of 30.8 Mb size encoding 8,317 proteins, and an additional completed mitogenome of 26 kb. This fully completed P. oxalicum genome will provide a supplemental resource for further gene functional research as well as interspecies or intraspecies comparative analyses.By using the WGS approach and different bioinformatics tools and methods, this work provides a deeper insight of the novel jkac300_Supplementary_DataClick here for additional data file.


PTEN tumor suppressor. In the present study, we investigated the causative gene of CS in a family of PTEN (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed AMBRA1 (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). AMBRA1 is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of AMBRA1 in the clinical manifestations of CS, we generated AMBRA1 depletion and Q30R mutation in hTERT-RPE1  using the CRISPR-Cas9 gene editing system. We observed that both AMBRA1-depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the AMBRA1 Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of AMBRA1 Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a situs inversus phenotype in ambra1-depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the AMBRA1 Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the  Cowden syndrome (CS), also known as multiple hamartoma syndrome, is a rare genetic autosomal dominant disorder affecting 1 in 200,000 people ,4. The mWe investigated the causative gene of CS in a family: the father and two daughters were diagnosed with CS, while the mother and one daughter were not affected. We performed whole exome sequencing (WES) analysis on all members of the family and identified AMBRA1 as the top candidate. AMBRA1 could be a good candidate from the genetic results, but its functional significance needs to be examined.AMBRA1, a key adaptor protein of the autophagy signaling network, is a tumor suppressor and mutated in a wide range of tumors ,7. In adIn our study, AMBRA1 Q30R mutant hTERT-RPE1 cells showed hyperproliferation, disturbed G1/S transition, and abnormal primary ciliogenesis. Primary cilia are immotile extracellular sensory organelles that are dynamically regulated during cell cycle progression. Primary cilia assemble in non-dividing cells of the G0/G1 phase and disassemble upon cell cycle entry . Therefohttps://j-rdmm.org/; accessed on 29 November 2018). As part of these projects, we aimed to identify the candidate gene for one CS family. The affected individuals of this study were the father and two daughters of the family (The IRUD (Initiative on Rare and Undiagnosed Diseases) and J-RDMM (Japan Rare Disease Models and Mechanisms Network) are ongoing multi-center research projects supported by the Japan Agency for Medical Research and Development  and R1195S (c.G3585C). Q30R, an AMBRA1 variant, exhibited the most deleterious effect according to the in silico pathogenicity prediction C, locate−/−)  entering the S and G2/M phases was significantly higher than that of the wildtype  . When wewildtype A,B, indipe cells C.AMBRA1 Q30R mutation affects cell proliferation similar to AMBRA1 depletion, we introduced the Q30R mutation in exon 2 of AMBRA1 to hTERT-RPE1 cells using the CRISPR-Cas9 gene editing system  which entered the G0 phase  which entered the cell division phases including G1 and S+G2/M   (To confirm the association of ambra1a depletion with the  0.0001) F. On thesitus inversus phenotype in ambra1a morphants but not in ambra1b morphants suggests that these two genes play different roles in zebrafish embryogenesis, with ambra1a being involved in the orientation of visceral organs, which could plausibly be through regulating primary cilia. This confirms that AMBRA1 function is tightly correlated with ciliogenesis. However, we could not observe any lesion similar to a hamartoma in the ambra1a knockdown zebrafishes.Our observation of the AMBRA1 Q30R as a novel candidate germline mutation of Cowden syndrome. The Q30R germline mutation, located in the highly conserved N-terminal region of AMBRA1, has not been documented previously. The N-terminal region of AMBRA1 is characterized by three WD40 domains which act as protein interaction scaffolds mediating the interaction of AMBRA1 with a wide range of proteins [In the present study, the overview of which is presented in proteins . Deletioproteins . This si+/−, −/−, Q30R A/G, Q30R G/G) confirmed the hyperproliferative potential of the AMBRA1 Q30 mutation, which could possibly be responsible for the benign self-limited growth of hamartomatous lesions in CS patients [+/− cells apparently showed a higher proliferation rate than the wildtype cells, which is consistent with previous reports of increased tumorigenesis in monoallelic AMBRA1 mouse embryonic fibroblast cells [AMBRA1 as a haploinsufficient tumor suppressor, sensitive to the gene dosage.Our observation of increased proliferation in AMBRA1-perturbed cells  and G1/S transition. We suggest that AMBRA1−/− cells could have lost G0/G1 transition control, while AMBRA1 Q30R mutant cells lost G1/S phase transition control. The mutants, AMBRAQ30R A/G and Q30R G/G, showing uncontrolled proliferation, albeit the unfavorable extracellular condition, signifies the pathogenicity and irreversible hyperproliferative ability of Q30R mutation and its possible influence on benign hamartoma and malignant transformation in CS patients.However, in nutrient-deprived conditions, wildtype and AMBRA1AMBRA1 depletion and AMBRA1 Q30R mutation could induce hyperproliferation through regulating cyclinD1, we analyzed cyclinD1 expression in AMBRA1+/−, AMBRA1−/−, AMBRAQ30R A/G and Q30R G/G cells  and the ciliogenesis phenotype. The heterozygous AMBRA1Q30R A/G cells showed ciliary assembly in nutrient-adequate conditions, and an irregular structure and multiciliation upon nutrient deprivation, while on the contrary, the homozygous AMBRA1Q30R G/G cells showed no ciliation. It is not uncommon for a single mutation in highly pleiotropic genes that are involved in more than one function and have multiple protein interactions to result in very different phenotypes depending upon the alleles involved [AMBRA1 Q30R and R1195S using AlphaFold2 demonstrated that both the mutations did not induce a significant structural change in the AMBRA1 tertiary structure . After participants gave informed consent, we performed whole exome sequencing using genomic DNA of both the affected and unaffected members of the CS family. The detailed procedure of WES is presented in the Supporting Information. We prioritized novel germline variants (i) based on their potential to alter protein structure or function  and (ii) those that were not reported in dbSNP and the 1000 Genomes Project, (iii) excluding common variants found in both the affected and unaffected family members and including germline mutations observed in all three probands, (iv) based on PhyloP, Polymorphism Phenotyping v2 (Polyphen-2) pathogenicity scores and GERP conservation scores. We predicted the structural changes induced by the missense variants (p.Q30R and p.R1195S) of our candidate gene AMBRA1 using AlphaFold2. Detailed protocol of the 3D-structural analysis is presented in the AMBRA1 Q30R editing (c.A89G), we designed a guide RNA -low glucose supplemented with 10% heat-inactivated fetal bovine serum and 1% Penicillin–Streptomycin. We created AMBRA1-depleted hTERT-RPE1 cells using the CRISPR-Cas9 system by deleting approximately 1093 bp between intron 6 and exon 7 . For AMBuide RNA  targetin10% PAGE . The AMB10% PAGE . To confquencing . Details+/− and AMBRA1−/− knockouts and AMBRA1Q30R A/G and AMBRA1Q30R G/G mutants in the cell cycle phases through flow cytometry using a BD Accuri flow cytometer  with Ki67 staining using an anti-Ki67 antibody  and DNA staining using PI. The fractions of cells in different cell cycle phases were calculated using FCS express software . We analyzed the distribution of AMBRA1+/− and AMBRA1 −/− knockout and AMBRA1Q30R A/G and AMBRA1Q30R G/G mutant cells in both nutrient-sufficient and starved conditions through immunocytochemistry using rabbit anti-arl13B primary antibody , a primary cilia marker. The immunostained cells were imaged under a Deltavision Personal DV fluorescent microscope . Quantification analysis of the nucleus and primary cilia was performed using Volocity 6.5 software . We observed the frequency of primary cilia in AMBRA1Detailed protocols of the flow cytometry and immunocytochemistry are presented in the ambra1 codes for two paralog nonredundant genes, ambra1a and ambra1b [ambra1 CRISPANTS and morphants were analyzed using a Leica M165 FC microscope . Details of the CRISPR/Cas9 and morpholino experiments on zebrafish are provided in the All zebrafish experiments were carried out with permission from the Committee for Animal Care and Use of the National Institute of Genetics, Japan . Adult zebrafish were maintained on a 13 h light/11 h dark cycle. Embryos were kept at 28° until processing for phenotypic analysis. In zebrafish,  ambra1b . We mani+/−, AMBRA1−/−, AMBRA1Q30R A/G, and AMBRA1Q30R G/G with the AMBRA1 wildtype cells and Fisher’s exact test to compare the situs inversus phenotype of ambra1a morphants with the wildtype zebrafish were performed using GraphPad prism V.9. .Statistical analyses such as ANOVA to compare AMBRA1