Introduction:Klebsiella pneumoniae (K. pneumoniae) and Pseudomonas aeruginosa (P. aeruginosa) are the most common Gram-negative bacteria associated with pneumonia and coinfecting the same patient. Despite their high virulence, there is no effective vaccine against them.Methods: In the current study, the screening of several proteins from both pathogens highlighted FepA and OmpK35 for K. pneumonia in addition to HasR and OprF from P. aeruginosa as promising candidates for epitope mapping. Those four proteins were linked to form a multitope vaccine, that was formulated with a suitable adjuvant, and PADRE peptides to finalize the multitope vaccine construct. The final vaccine’s physicochemical features, antigenicity, toxicity, allergenicity, and solubility were evaluated for use in humans.Results: The output of the computational analysis revealed that the designed multitope construct has passed these assessments with satisfactory scores where, as the last stage, we performed a molecular docking study between the potential vaccine construct and K. pneumonia associated immune receptors, TLR4 and TLR2, showing affinitive to both targets with preferentiality for the TLR4 receptor protein. Validation of the docking studies has proceeded through molecular dynamics simulation, which estimated a strong binding and supported the nomination of the designed vaccine as a putative solution for K. pneumoniae and P. aeruginosa coinfection. Here, we describe the approach for the design and assessment of our potential vaccine. Klebsiella pneumoniae is a Gram-negative bacteria that cause hospital and society-induced infections all over the world  and P. aeruginosa (strain ATCC 15692/DSM 22644/CIP 104116/JCM 14847/LMG 12228/1C/PRS 101/PAO1) were downloaded from the UniProt webserver (https://www.uniprot.org/) under the proteome ID of UP000000265 and UP000002438, respectively. As mentioned in the introduction section, the current study aims to design an epitope-based vaccine through the filtration of protein candidates belonging to the outer membrane and iron uptake proteins. Therefore, we selected nine K. pneumoniae protein candidates namely FepA, FepB, FepC, FhuA, FhuF, FuR (iron uptake proteins), OmpA, OmpC, and OmpF (outer membrane proteins), and filtered them through their antigenicity score estimated by VaxiJen v2.0 , OprF, and OprH (outer membrane proteins) were filtered and 2 proteins  namely HasR and OprF with the antigenicity scores of 0.59 and 0.8 respectively were selected as our final candidates for P. aeruginosa.The reference proteomes of Jen v2.0  with theThe filtered 4 proteins from the previous step were uploaded to SignalP- 5.0 server  to prediin vivo separation of the joined epitopes  was utilized.While continuous B cell epitopes, which were predicted in the above sections, are estimated through the primary amino acid sequence of a protein, another type of epitopes, which is conformational (or discontinuous) B cell epitopes, are predicted based on the 3D structure of the antigenic protein. For this purpose, the ElliPro Server  (https://cluspro.org/)  and TLR4 (PDB: 3FXI) and constructed hybrid multi-epitope vaccine were independently prepared through partial charge assignment, 3D-protein protonation and/or removal of any bound ligand(s), crystalized solvent, and ionic metals/salts . For ide/CPORT/) . Dockingro.org/) . Relyingro.org/) . Interacro.org/) . It was ro.org/) .https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver) for macromolecular interface analysis . The last two descriptors are indices for higher interface hydrophobicity/protein affinity  and to how many degrees the protein-protein interface can be interaction-specific (p < 0.5) (http://cadd.zju.edu.cn/hawkdock/)  . Estimatwkdock/) . HawkDocwkdock/) .via sufficient 0.15 M sodium and chloride ions. Systems were subjected to the steepest-descent minimization at 0.05 ns, followed by two-staged equilibration at standard thermo- and barostats , and RMS-fluctuations  relying on the protein’s backbone alpha-carbon atoms. Conformational analysis and visualization of the simulated complexes at specified timeframes were done using PyMOL software .The best-docked vaccine-TLR complex was subjected to 100 ns all-atom molecular dynamics simulations under CHARMM36m forcefield and GROMACS program . Proteineraction . Trajectvia the injection of the potential vaccine three times at 4 weeks intervals. This approach has been followed to get a long-lasting immune response.The final stage of our study was the prediction of the generated immune response upon the administration of the potential vaccine construct where The C-ImmSim server  was emplE. coli k-12 strain as the expression organism as it is frequently used in gene cloning experiments . The codon adaptation index (CAI), a value that is calculated by the server, gives an estimation for the constructed potential vaccine to be expressed in E. coli k-12.The last stage of the current study was the codon optimization for the designed potential vaccine where we employed the JCAT server for this purpose . Here, wK. pneumoniae, 9 protein candidates namely FepA, FepB, FepC, FhuA, FhuF, FuR (iron uptake proteins), OmpA, OmpC, and OmpF (outer membrane proteins) were filtered through their antigenicity score estimated by VaxiJen v2.0 with the cutoff score of 0.4  and the final 2 protein candidates of K. pneumoniae were FepA and OmpF with antigenicity scores of 0.76 and 0.81 respectively (highest scores). Moving to P. aeruginosa, the same approach was followed where six protein candidates namely FoxA, FpvA, HasR, HitA (iron uptake proteins), OprF, and OprH (outer membrane proteins) were filtered and 2 proteins  namely HasR and OprF with the antigenicity scores of 0.59 and 0.8 respectively were selected as our final candidates for P. aeruginosa. Collectively, 4 proteins were selected as final targets for epitope prediction (2 proteins per microorganism).Regarding At this stage, the 4 filtered protein candidates were mapped for their T and B cell epitopes. For T cell epitopes, the mapping of MHC-I epitopes resulted in a large number of epitopes for each protein with a percentile rank ranging from 0.01 to 100 where a small percentile rank represents a better binding in comparison to the large percentile rank score, and for this reason, we filtered only the epitopes with percentile score less than 1 (to guarantee for good binding) and the best candidates with a large antigenicity score were listed in As the next stage after epitope mapping, we planned to select the top 2 single epitopes  from our final list of MHC-I, MHC-II, and B cell epitopes and after selecting the most promising B and T cell epitopes, we get a final count of 8 CTL, 8 HTL, and 8 BCL epitopes. These epitopes were linked together using GGGS, GPGPG, and KK linkers respectively. Following that, 2 important components, namely PADRE peptide and β-defensin, were incorporated to finalize the sequence of the multitope construct which lastly constituted 476 amino acids and sequenced as the following:“EAAAKGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKKEAAAKAKFVAAWTLKAAAGGGSKQEPKKYNYGGGSQTNPNYILYGGGSRTNGVATYRGGGSDTSSDDTTYGGGSSVDGMRQNYGGGSRVKHSPVAYGGGSKVHGNLTSLGGGSKSKVKENSYGPGPGLVRWEFAPMQSLEFEGPGPGIIPEYTLNSTLSWQVGPGPGFFGLVDGLSFALQYQGPGPGAVVQYQFDFGLRPSIGPGPGVSQDDLVQMSPSVISGPGPGDLSTLRANYGLEFFYGPGPGGAGLKYYFTENFFAKGPGPGGVGLRPYVSAGLAHQKKGNSTSGQRGNNKKAPEHKDEGSTKRTKKDASNVEGSQTTKKKSNRSVDQKADGNGDKAKKDSASQQQTALRRVRLDIKKSGLGGDANGTKKNITNINSDSQGRQQMTKKSVGTDAYNQKLSERKKAKFVAAWTLKAAAGGGS”.As mentioned in the methodology section, T cell epitopes mapping was performed against the HLA allele reference set to achieve a high percentage of population coverage, consequently, assessment of the percentage of population coverage revealed the scores of 92.56%, 99.98%, and 100% for world MHCI, MHCII, and combined population coverage respectively . Prior tIn order to assess the chemical properties of the potential multitope vaccine it was essential to predict its tertiary structure and for this purpose, we employed the Robetta webserver. The generated 3D model was then uploaded to the GlaxyRefine webserver where Ramachan plot analysis and ProSa Z-score were utilized to assess the quality of the original model generated by Robetta and the refined one. For the current study primary model (output of Robetta prediction), 85.3%, 13.4%, and 1.4% of residues were located in favored, allowed, and outlier regions, respectively. After refinement on the GlaxyRefine server, the refined model demonstrated 89.9%, 10%, and 1.1% of residues located in favored, allowed, and outlier regions, respectively with a Z-score of −4.59. The predicted tertiary structure of the multitope construct and the output of its structural validation are shown in The generated 3D structure of the multitope construct was uploaded to EIIiPro webserver for the prediction of conformational B cell epitopes. The server estimated 9 discontinuous epitopes  with a sK. pneumonia  for the obtained vaccine towards TLR4 over those at TLR2  . Besides polar interacting residues, hydrophobic interface residues of the multiepitope vaccine showed significant closeness and relevant non-polar contacts with neighboring TLR4 amino acids. Vaccine hydrophobic residues including; Trp181, Phe183, Tyr202, Leu208, Phe227, Tyr231, Tyr242, Phe244, Tyr286, Leu288, Tyr292, Tyr303, Phe305 depicted ≤5.0 Å distances from Met41, His229, His256, Phe377, Tyr403, Tyr451, His426, Ile450, Phe408, Phe500, and/or Val602 of the TLR4 interface, respectively.Key binding residues at the interface between the vaccine and each TLR target were also highlighted d Asp84) . AdditioGlu182, was the most frequent amino acid contributing to the vaccine’s parallel β-sheet stability towards the TLR2 concave interface. Other polar vaccine residues mediated significant hydrogen bonding with corresponding TLR2 amino acids at close distances and relevant angles (1.65 to 2.35 Å and 141 to 179 Å). Interestingly, the peptide backbones of several vaccine’s hydrophobic residues including; Trp62, Pro185, Leu189, Gly195, Ile198, Pro200, Leu288, and Tyr324, depicted relevant polar contacts with close TLR2 residues at the binding interface. In addition to hydrophilic residue pairing, hydrophobic interface residues of the multiepitope vaccine showed significant closeness and relevant non-polar contacts with neighboring TLR2 amino acids. Vaccine’s hydrophobic residues such as; Phe58, Val59, Trp181, Phe183, Met186, Phe191, Ile199, Leu208, Trp210, Leu225, Phe227, Leu229, Ala238, Val240, Tyr242, Phe246, Tyr286, Phe290, Tyr292, Leu301, Tyr302, Phe305, Val325, Ala327, and Leu329 showed distances less than 5.0 Å from Tyr109, His202, Ile204, Leu250, Ile251, Leu280, Tyr364, Leu392, Ile393, Tyr440, Ile461, Tyr483, Met500, Leu502, Val503, Trp529, His 531, and/or Pro568 of the TLR4 side.Moving towards the vaccine-TLR2 docking complex, several polar contacts were depicted majorly through hydrogen bond contacts without relevant salt bridges . The vacArg17–11.06 Kcal/mol, Tyr231–9.01, Asp60–7.18 Kcal/mol, Asp84–7.01 Kcal/mol, and Glu154–5.71 Kcal/mol) conferring their significant role for vaccine binding and TRL4-associated complex stability. Similarly, the vaccine’s polar residues; Glu182–8.49 Kcal/mol, Gln187–7.01 Kcal/mol, and Ser326–6.82 Kcal/mol, as well as those of TLR2 interface; Cys226–5.51 Kcal/mol, Asp366–4.28 Kcal/mol, and Glu310–3.79 Kcal/mol, were assigned with the high binding energy contributions.Putting all the above preferential per-residue interaction within energy terms, the MM/GBSA binding energy calculations were estimated for both docked vaccine/TLR complexes. As expected, higher residue-wise energy contributions were assigned for the key interacting residues of both vaccine and TLR proteins . The higThe RMSD trajectories of the multitope vaccine, TLR proteins, and their respective complex were monitored across the 100 ns all-atom simulation runs in reference to the alpha-carbon atoms (αC) of their respective initial structures. All monitored RMSD tones showed a gradual increase for the first 25 ns, while after that RMSDs started to attain steadier trajectories till the end of the simulation runs . The latC-terminal residues and vicinal amino acid region . On the other hand, the vaccine’s N-terminal residues were of relevant stability and lower RMSF values. However, single residue-range Thr100-Gly162 depicted the highest fluctuations at the TLR4-bounded system (RMSF up to 13.03 Å) and even much higher flexibility at the TLR2-bounded one (RMSF up to 19.87 Å). It is worth noting that both vaccine’s side high-fluctuating residue regions were parts of the vaccine that are greatly extended towards the solvent side, quite distal from the TLR binding interface. Regarding the inherited fluctuations of the simulated TLR proteins, RMSF tones were at regular fluctuation patterns resembling repetitive camel humps. Interestingly, typical protein dynamic behaviors were depicted for simulated TLRs since their core residue ranges were assigned with lower RMSFs than those at the terminal sides. The observed differential fluctuation patterns between the vaccine and/or TLRs would question the influence of the protein’s inherited ternary structure, dimerization interface, and vaccine binding on protein simulation patterns.Dissecting the protein fluctuation pattern down to their respective residues, the αC-RMSF trajectories were monitored across the simulation run for each simulated protein, including the vaccine and TLR protomeric unit . NotablyTwo other trajectory-based stability parameters, ROG and SASA, were also monitored across the simulation runs. In relation to their respective central masses, both simulated multitope vaccines showed differential ROG values across different simulation times . Bound t2) for each simulated complex was estimated by adopting the sole SASAs of each bounded protein (SASAvaccine and SASATLR) in addition to the entire bounded complexes; BSA = 0.5*(SASAvaccine + SASATLR – SASAcomplex)   . The sim7.73 Å2) . NotablyN-terminus. In the TLR4-bound complex, the latter vaccine’s high-flexible regions adopted more compacted conformations as they became more directed toward the lateral interface of the TLR4 protein at the end of the simulation time. On the contrary, the vaccine’s flexible regions at the TLR2 complex could not manage to adopt similar compactness the thing that was translated into higher distortions for the vaccine’s C-terminal side. Such dynamic behavior could explain the higher RMSF-related fluctuation patterns associated with these solvent-exposed residue regions as well as the higher RMSD/Rg tones for TLR2-associated vaccine over that at the TLR4 interface. The rest of the vaccine ternary structures at both TLR complexes showed limited conformational changes, particularly those being anchored deep at the TLR binding interface (inner concave surface).Conformational analysis through aligning the starting and final complex structures illustrated differential orientations for the bound ligand at the TLR binding interface . ObserveTotal = −10804.19 ± 138.19 kJ/mol versus −2,454.162 ± 410.95 kJ/mol) (Electrostatic) was depicted over the van der Waal interactions (ΔGvdw). The electrostatic potential energy contributions were more than 3-folds and 12-fold that of the van der Waal interactions at TLR4 and TLR2 interfaces, respectively. Notably, the polar solvation penalties (ΔGPolar solvation) at both TLR2 and TLR4 systems were almost comparable and were translated as repulsive forces against vaccine binding as well as compromised complex stabilities since binding is a solvent-displacement process (SASA and ΔGvdw) for the TLR4-associated complex over the TLR2 would have managed to overcompensate these unfavored repulsive polar solvation forces. All these furnished binding energy terms were translated into better vaccine affinity towards TLR4 over the TLR2 side. Finally, the relatively high total non-polar potentials could be correlated to the large hydrophobic receptor interface available for the multitope vaccine binding.Moving towards the vaccine-TLR binding affinity across the simulated thermodynamic trajectories, the MM_PBSA calculations revealed great concordance with the preliminary HawkDock-MM/GBSA docking binding scores. The trajectory-based MM_PBSA revealed higher negative average free binding interaction energies for the vaccine towards the TLR4 receptor interface as compared to those at the TLR2 side (ΔG kJ/mol) . At both process . NeverthN-terminal residues as well as those constituting the extensive anti-parallel sheets. Repulsive positive-value energy contributions were limited to the residue area being exposed towards the solvent side, particularly C-terminal residues and vicinal amino acid region. Findings were consistent with the above-depicted RMSF values showing residues with high positive-value energy contributions to be correlated with high respective RMSF trajectories across the simulation time. Concerning the protein receptor, higher negative-value residue-wise energy contributions and lower repulsive positive contributions were assigned for TLR4 over the TLR2 unit. On the other hand, preferentially higher negative-value energy contributions were assigned for the N-terminal half of each TLR structure.Exploring the residue-wise free binding energy contributions for the simulated complex were highlighted in Figures 6B, C. Higher negative-value energy contributions were assigned for the vaccine The computational assessment through the C-ImmSim server predicted an overall satisfactory response with the successive doses of the potential multitope vaccine injection . It is cE. coli k-12 .The output of the JCAT servers showed that the GC content of the improved sequence was 51.6% . Regarding K. pneumoniae, FepA and OmpF were selected as our final targets for epitope mapping. FepA is an integral outer membrane protein that is composed of 742 amino acid residues and transports ferric enterobactin where several reports revealed that immune responses against FepA can inhibit K. pneumoniae infections  therefore Robetta has been and continues to be among the most consistent top-performing servers and consequently, it has been employed in the 3D structure prediction of multitope constructs in several similar studies (The approach of computational vaccine design has been employed in several previous trials where some reports were only interested in the nomination of potential vaccine candidates  or startng stage . The curng stage . For B cng stage . The cur studies .in vivo (+ T cell responses and potentiate the immune response, respectively (After the filtration steps of the protein candidates, the selected ones were mapped for their B and T cell epitopes. Reliance on the epitopes for the vaccine design, instead of the whole protein, provides an important advantage of targeting only the antigenic parts of the protein and lessening the probability of allergic reactions . On the in vivo . Startinin vivo . Additioectively . FollowiK. pneumoniae-directed host defenses (K. pneumoniae-host disseminations were associated with TLR4 and TLR2 immune responses within the pneumonia mouse model (K. Pneumoniae, Dar et al. reported successful affinity for their immunoinformatics-designed multiepitope vaccine towards the inner concave interfaces of both TLR4 and TLR2 (via its highly packed anti-parallel β-sheets ternary structure the thing that was adopted throughout the relevant selection of the docked binding mode.Accumulated evidence correlated the important role of both TLR2 and TLR4 immune receptors within the defenses . Both rese model . While Tse model . In our se model . The innse model . Focusinand TLR2 . Notablyα-helices with flexible β-loop connections within the vaccine’s designed structures could reason for higher inherited flexibility throughout the simulation runs. Additionally, the initial docking pose with an extended C-terminal would favor a great conformational shift for final convergence into more stable compacted conformations. On the contrary, the densely packed TLR orchestra with shoe-like structure would advent from its several highly ordered parallel β-sheets the thing that would be correlated with limited flexibility and thermodynamic fluctuations. Comparable flexibility patterns were also depicted within several reported studies investigating the potential binding affinity of peptide-based vaccines toward microbial TLRs (K. pneumoniae strains (The preferential binding of multitope vaccine to the TLR target was demonstrated through a multi-level stability analysis. The RMSD analysis was significant for showing limited conformational changes and superior relative stability depicting steady tones across the simulation times. On general bases, RMSD trajectories provide accurate measurement regarding a molecular deviation from its reference structure at the beginning of the molecular dynamics simulations . High prial TLRs . Compara strains .Stability patterns were successfully translated into high negative free binding energy. Both MM_GBSA and MM_PBSA binding energy calculations for respective docked and molecular dynamics complexes illustrated the predominance of electrostatic potentials and polar residue energy contributions for the vaccine towards the TLR site. Greater electrostatic negative values, suggesting stronger binding affinities, were consistent with reported results of other research groups investigating vaccines of other microorganism origins towards different TLRs, including our investigated ones TLR4 and TLR2 . It is win silico evaluate potential vaccine candidates is a promising technique that witnessed great development in the last few years. With the advantages of being a time and cost-saving approach, we selected 4 protein candidates from the proteome of K. pneumoniae and P. aeruginosa and mapped B and T cell epitopes for these candidates. The most promising mono epitopes were integrated into a multitope construct which in turn was evaluated for its physicochemical, immunological, and binding characteristics with 2 TLRs. Results of the computational assessments nominated our multitope construct as a potential vaccine against the coinfection status of K. pneumoniae and P. aeruginosa. Future wet lab validation is an essential next step to validate the current findings.The continuous increase in the rates of bacterial antibiotic resistance makes it a public health concern to develop novel solutions. Reliance on the computational tools to design and


Axial spondyloarthritis (axSpA) is a common chronic inflammatory disease, associated with extracellular matrix (ECM) remodeling of the cartilage, bone, and connective tissues. The primary symptom of axSpA is back pain, caused by inflammation. However, there is a medical need to truly identify patients with axSpA from other subjects with buttock or low back pain attributable to other reasons. We aimed to investigate circulating biomarkers of ECM/inflammation (MMP-degraded type I (C1M), II , III (C3M), IV (C4M), VI (C6M), and X  collagens, CRPM, PROM and VICM) and ECM formation of type II (PRO-C2), III (PRO-C3), IV (PRO-C4), and VI (PRO-C6) collagens as potential biomarkers to identify patients with axSpA.N = 41), women with postpartum buttock/pelvic pain (N = 46), disc herniation (N = 25), and a group of healthy subjects (including women without postpartum pelvic pain (N = 14), subjects with various types of physical strain (cleaning staff (N = 26) long-distance runners (N = 23)), and healthy men (N = 29)). Differences between the groups were calculated by ANCOVA and AUC, while Spearman’s correlations were performed with ECM biomarkers and clinical scores.We measured biomarkers from a cross-sectional study with 204 participants by enzyme-linked immunosorbent assay (ELISA). The study included axSpA patients (p < 0.05-p < 0.0001) compared to all the non-axSpA control groups. Further, C6M and PRO-C4 were significantly higher in patients with axSpA (both p < 0.0001) compared to women with postpartum pelvic pain and healthy subjects, whereas PRO-C3 was significantly lower compared to healthy subjects (p = 0.01). The best ECM common biomarker to differentiate between axSpA and the non-axSpA control groups was PRO-C4 . Mild correlations were observed between collagen turnover and inflammation biomarkers and CRP and MRI .Patients with axSpA expressed significantly higher levels of C1M, C4M, and VICM (Biomarkers of type I, IV, and VI collagen and biomarkers of inflammation showed an altered turnover in patients with axSpA compared with the non-axSpA control groups. Such biomarkers may be useful in combination with MRI or independently to separate patients with axSpA from other back pain conditions.The online version contains supplementary material available at 10.1186/s13075-022-02839-1. Axial spondyloarthritis (axSpA) is a chronic inflammatory rheumatic disease that predominantly affects the axial skeleton. The clinical characteristics defining axSpA are chronic back pain and sacroiliitis displayed on magnetic resonance imaging (MRI), and with time, patients may get progressive ankylosis of the sacroiliac (SI) joint and spine leading to ankylosing spondylitis (AS) . AxSpA gThe extracellular matrix (ECM) is formed by collagens, elastin, laminin, fibronectin, and proteoglycans. Type I, II, III, IV, and VI collagens are the main structural elements of the ECM . ProlargWhen proteases degrade the ECM, metabolites are generated and detection of those that are derived in the specific/local tissue can reflect local pathology and be quantified in blood . MetabolIn this study, we investigated the circulating ECM biomarkers and their potential to differentiate axSpA patients from subjects with or without buttock or pelvic pain attributed to other reasons, particularly post-partum women, as well as a large group of healthy subjects. In addition, we aimed to investigate the association of ECM-derived biomarkers with clinical parameters and whether these biomarkers could separate axSpA from non-axSpA controls.N = 41), (2) women with postpartum buttock/pelvic pain 4 months to 16 month after delivery (N = 46), (3) women without postpartum buttock/pelvic pain 4 months to 16 months after delivery (N = 14), (4) patients with lumbar disc herniation (N = 25), (5) cleaning staff (N = 26), (6) long-distance runners (N = 23), (7) a group of healthy men (N = 29). Cleaning staff and long-distance runners were included to have a group which had repeated strain in daily life. All participants signed informed consent before study inclusion. The study was approved by the local ethical committee  and conducted in accordance with the Declaration of Helsinki V and Danish legislation.A total of 204 participants were included in the MASH study—a prospective, cross-sectional study conducted at Rigshospitalet, Copenhagen, Denmark, from 2013 to 2016 . The MASDemographic and clinical data were acquired from all participants. Participants were evaluated for exclusion criteria (described in ) to ensuA panel of ECM turnover biomarkers was measured in serum using validated ELISAs and IDS automated chemiluminescent assay.We measured the biomarkers: MMP-degraded type I C1M) , II  value ≥ 0.3 or ≤ − 0.3 were included. An area under the receiver operating characteristic curve (AUC) was used for exploring the separation potential of the metabolites among patients with axSpA and non-axSpA independently. Data analyses were performed using R studio version 4. 0. 3 . Graphical illustrations were created using GraphPad Prism version 9.00 for Windows .Baseline characteristics are described as number (frequency) and percentage for categorical variables, and as mean (± SD) for continuous variables. Kruskal-Wallis rank test was used to examine baseline differences between axSpA patients and the other groups of participants. The biomarker data was natural log-transformed for normalization and four new ratios of degradation/formation of type II (C2M/PRO-C2), III (C3M/PRO-C3), IV (C4M/PRO-C4), and VI (C6M/PRO-C6) collagen were also included and natural log-transformed. Differences in the biomarker levels and type II, III, IV, and VI collagen degradation/formation ratios among patients with axSpA and each of the other groups were examined by ANCOVA analyses adjusting by confounders. A nominal significance level of 5% was used for the analyses and adjustment for multiple comparison were performed within each biomarker, by Tukey method, but not across biomarkers. Spearman’s correlation test was used in analyses between the biomarker levels and clinical assessments in the patients with axSpA, women with postpartum pain, and patients with disc herniation. Only biomarkers showing one or more correlations with a rho , MMP-driven citrullinated vimentin degradation , type IV collagen formation , and degradation/formation ratio of type VI collagen  compared to women with buttock/pelvic postpartum pain when adjusting for confounders age, gender and body mass index (BMI)  when compared to patients with disc herniation  and significant decreased levels of type III collagen formation . Similar significant differences were observed when further adjusting for the participants’ physical activity (from light to most heavy) or their smoking status (from daily smoker to never smoked) (Table p < 0.05 in both) and type IV degradation/formation ratio (C4M/PRO-C4) was significantly lower in patients with axSpA compared to patients with disc herniation (p < 0.05). However, when further adjusting for MRI inflammation score, no significant differences were found in C4M or VICM among patients with axSpA vs. women with pelvic postpartum pain, vs. patients with disc herniation or vs. healthy controls  Table . Patienton Table . Furtherp < 0.01 for all except VICM (p < 0.05)), and the C3M/PRO-C3 ratio was also significantly increased (p < 0.001). C1M, C6M, PRO-C4, and VICM showed significantly increased levels in the axSpA group compared to the healthy men group. PRO-C4 also showed higher levels in the axSpA group compared to the cleaning staff group and the women with pelvic postpartum pain group . C6M/PRO-C6 ratio showed significantly higher levels in the group of patients with axSpA compared to the women with pelvic postpartum pain (p < 0.01). Neither C2M, T2CM, PRO-C2, C2M/PRO-C2, C4M/PRO-C4, nor PROM showed any significant differences between the axSpA group and each of the other groups; see Figure S1.We also explored for differences between patients with axSpA and each of the initial seven groups , whereas Bath Ankylosing Spondylitis Metrology Index (BASMI) showed a mild to moderate correlation with C6M and C6M/PRO-C6 . From the MRI SI Joint Structural Scores (SSS), erosion presented a mild correlation with C10C, and C6M/PRO-C6  and a moderate to high correlation with PRO-C6 . SSS Backfill score presented a mild to moderate correlation with T2CM, C10C, and PRO-C4 , whereas SSS ankylosis score was mild-moderately correlated with C3M, C4M, C6M, PROM, C3M/PRO-C3, C4M/PRO-C4, and C6M/PRO-C6 .In patients with axSpA, PRO-C3 and C3M/PRO-C3 mild–moderately correlated with age , tender joint count (TJC), SPARCC SSS fat score, and SSS erosion score . C4M, C6M, and PROM were mildly correlated with CRP , whereas PRO-C4 presented a moderate correlation . COL10NC, PRO-C6, CRPM, and the ratio C6M/PRO-C6 presented a mild-moderate correlation with symptom duration . C10C, PRO-C4, and C4M/PRO-C4 were mild–moderately correlated with BASMI , and only C3M presented a mild correlation with inflammation score . C3M, C6M, PRO-C4, PRO-C6, CRPM, C10C, COL10NC, C4M/PRO-C4, and C6M/PRO-C6 did not show any relevant correlation with age (Table S1). C1M, C2M, T2CM, VICM, PRO-C2, PRO-C3, C2M/PRO-C2, and C3M/PRO-C3 did not present any relevant correlation with the clinical parameters.We further examined the correlation between ECM metabolites and clinical parameters in the women with pelvic postpartum pain and in the patients with disc herniation . From the clinical variables, we could include age, BMI, CRP, symptom duration, BASMI, and SPARCC SI joint inflammation score. In women with postpartum pain, C6M, PRO-C4, and the ratio C4M/PRO-C4 presented a mild correlation with BMI , whereas CRPM and the ratio C4M/PRO-C4 presented a mild correlation . CRPM also presented a mild correlation with BMI . C1M and PROM showed a mild correlation with CRP , whereas PRO-C4 and C3M/PRO-C3 showed a moderate correlation . PRO-C4 mildly correlated with symptom duration , whereas PRO-C6 showed a moderate correlation . C2M mildly correlated with BASMI , and together with PRO-C3, it showed a mild to moderate correlation with SPARCC SI joint inflammation score . C1M, C2M, T2CM, COL10NC, VICM, PRO-C2, C2M/PRO-C2, and C6M/PRO-C6 did not present any relevant correlation with the clinical parameters.In patients with disc herniation, C4M presented a moderate correlation with age , and healthy men. The data suggest an increased rate of type I, IV, and VI collagen degradation , type III and IV collagen formation (PRO-C3 and PRO-C4), and chronic inflammation (VICM) in axSpA patients compared to women with postpartum pelvic pain, patients with disc herniation, and healthy subjects. The biomarkers C1M, C3M, C4M, C6M, PRO-C4, and CRPM moderate–strongly correlated with CRP. The AUC analysis showed that the overall best ECM biomarker to separate patients with axSpA from both women with postpartum pain and healthy subjects was PRO-C4. To our knowledge, this is the first study to use ECM turnover biomarkers to identify axSpA patients from women with postpartum pelvis pain and other non-axSpA subjects suffering from similar symptoms, and this is important because MRI detected bone marrow edema (BME) in the SI joints is frequent in these groups, where one or more ECM biomarkers potentially may have a clinical utility. These results demonstrate that collagen turnover and inflammation biomarkers derived from the ECM are upregulated in serum from axSpA patients and may indicate disease activity. Such biomarkers may have a future role as diagnostic tools for axSpA together with MRI or potentially independently. In addition, these ECM biomarkers may be useful to separate patients with axSpA from other back pain conditions that challenge diagnostics in routine care.Recently, early diagnosis has been improved due to advances of MRI techniques; however, it is costly, not widely available, and requires experienced professionals to interpret the findings . MoreoveWe explored a panel of biomarkers to identify the best ECM biomarker for differentiation of patients with axSpA from subjects suffering from other conditions with low back pain or healthy subjects with various types of strain. The levels of C1M, C4M, and C6M were higher in axSpA patients compared to women with postpartum pelvic pain, patients with disc herniation and healthy controls, indicating an enhanced ECM turnover of soft tissue and joint structures. Specifically, C1M and C6M indicate a turnover of the connective tissue, whereas C4M suggests the remodeling of the basal lamina . These rRemodeling of the ECM involves the formation of new collagens in the interstitial connective tissue matrix and the basement membrane . We founChronic inflammation is one of the hallmarks of axSpA . The bioIn the current study, clinical data such as symptom duration, clinical indices, and MRI of the SI joints were available and allowed evaluation of ECM turnover biomarkers with disease activity and MRI measurements of inflammation and structural damage. No strong correlations were found between the ECM turnover biomarkers and the clinical data or MRI findings in patients with axSpA. However, C3M and PRO-C3 showed a mild correlation with symptom duration of the disease in patients with axSpA, and C3M presented a mild-moderate correlation with the total SPARCC inflammation score in women with pelvic postpartum pain.The SPARCC SSS erosion score presented a mild to moderate correlation with C10C and PRO-C6, the backfill score a mild to moderate correlation with T2CM, C10C, and PRO-C4, and the ankylosis score a mild to moderate correlation with the ECM turnover biomarkers C3M, C4M, C6M, and PROM in patients with axSpA. Erosion, backfill, and ankylosis scores reflect bone destruction, early bone formation, and late bone formation, respectively  showed aCRP presented the best diagnostic capacity to differentiate patients with axSpA from women with pelvic postpartum pain, patients with disc herniation, and healthy controls, while PRO-C4 was the best ECM turnover biomarker for segregating patients with axSpA from women with pelvic postpartum pain and healthy controls. In contrast, Hušáková et al.  and Bay-The major strength of this study was the heterogeneity in the non-axSpA control group, including subjects with and without back pain. Moreover, all the clinical data regarding the MRI of SI joints obtained from all the subjects of the study allowed us to evaluate correlations between the biomarkers data and local inflammation. Limitations of the study include the sample size and an uneven gender distribution between the groups. Another limitation is the lack of erythrocyte sedimentation rate (ESR), since this has been shown to reflect inflammation in patients with axSpA and would be interesting to compare with the ECM biomarkers .In conclusion, this study showed an altered ECM turnover quantified by biomarkers of type I, III, IV, and VI collagen and inflammation in patients with axSpA compared to non-axSpA subjects. Some associations between the biomarkers and clinical data and MRI scores, respectively, were found. PRO-C4 was the best ECM biomarker for separating patients with axSpA from non-axSpA subjects, but further studies are needed to identify biomarkers as true diagnostic tools. Overall, the investigated biomarkers may potentially be useful, either in combination with MRI or independently, to separate patients with axSpA from other back pain conditions; however, further validations are required.Additional file 1: Supplementary Figure S1. Biomarker levels compared between groups. Table S1. Correlations of the biomarkers with clinical variables in women with pelvic postpartum pain. Table S2. Correlations of the biomarkers with clinical variables in patients with disc herniation.


However, the various mechanisms of dynamic m5C deposition and removal, and the role m5C plays within the cell, remains to be fully understood.DNA and RNA methylation dynamics have been linked to a variety of cellular processes such as development, differentiation, and the maintenance of genome integrity. The correct deposition and removal of methylated cytosine and its oxidized analogues is pivotal for cellular homeostasis, rapid responses to exogenous stimuli, and regulated gene expression. Uncoordinated expression of DNA/RNA methyltransferases and demethylase enzymes has been linked to genome instability and consequently to cancer progression. Furthermore, accumulating evidence indicates that post-transcriptional DNA/RNA modifications are important features in DNA/RNA function, regulating the timely recruitment of modification-specific reader proteins. Understanding the biological processes that lead to tumorigenesis or somatic reprogramming has attracted a lot of attention from the scientific community. This work has revealed extensive crosstalk between epigenetic and epitranscriptomic pathways, adding a new layer of complexity to our understanding of cellular programming and responses to environmental cues. One of the key modifications, m Three ding RNAs . Epigeneing RNAs . The dyning RNAs , transpoing RNAs , and braing RNAs . Furthering RNAs   , methyla-IP-seq)  and TET--IP-seq) , were sud ncRNAs . Althougreported . A largereported . Oncogenreported . Furtherreported . In thisde novo DNA methylation  proteins, which exist as 3 isoforms: TET1, TET2, TET3 , alters the affinity of DNMT1 for the methylated site, and results in loss of the epigenetic mark over several rounds of DNA replication. RNA m5C modification has been found in mRNAs, rRNAs, tRNAs, and ncRNAs. The RNA specific subset of S-adenosyl-methionine-dependent methyltransferases includes TRDMT1 (tRNA aspartic acid methyltransferase 1)   , also knC on RNA . The m5Ctal cues . For exar export . Changes therapy . Furtherspecific . The prefidelity . In tRNA (47–50) . The preof genes . In mRNA regions . m5C hasult RNAs , and its5C-driven epigenetic and epitranscriptomic events are entwined in cellular homeostasis. Deregulated m5C deposition can promote cancer development as a consequence of deregulation of both DNA and RNA molecules  have been described to be essential during DNA damage repair  proteins are considered m5C erasers. Active demethylation of m5C is achieved by TET-mediated sequential oxidation with the production of hm5C (5-Hydroxymethylcytosine), f5C (5-formylcytosine), ca5C (5-carboxylcytosine) analogues (5C and ca5C is processed by the Thymine DNA Glycosylase (TDG) to form abasic sites, followed by Base Excision Repair (BER) to restore the unmodified cytosine (5C) and decarboxylation (ca5C) mediated by DNMT3A/B  AID/DNMT3A/B , and 3) DNMT3A/B . However3A/B   protein family plays a pivotal role in determining the transcriptional state of the epigenome and shows a strong preference for hm5C over the m5C modification  . Furtherare hm5C . The preolecules . m5C andfication . MBD pro5C and ca5C oxidized forms of m5C are considered short-lived intermediates in the demethylation process, and their steady-state levels are many orders of magnitude lower than hm5C, emerging evidence indicates that f5C and ca5C might have independent epigenetic signalling roles in recruiting modification-specific “reader” proteins  differentiation (5C deposition drives site-specific demethylation of reprogramming enhancers and promoters resulting in the formation of iPSC (induced pluripotent stem cells)  differentiation . Cell farocesses . The RNAy manner . TET-depntiation . During m cells) . TET trignatures .5C has been proposed to act as a novel diagnostic and prognostic marker in several human malignancies   . Severalopoietic . The dersistance .5C accumulation has also been described at DNA damage, suggesting a crucial role for hm5C in promoting DNA damage repair  in vitro and in vivo  (1A) (7G) (4C)   and FTO and ALKBH5 enzymes, respectively , can be explained by the involvement of specific m6A readers in the stability of either oncogene or tumor-suppressor m6A-modified mRNAs  , N1-meth6A) (1A) , N7-meth1A) (7G)  and N4-m7G) (4C)  have bee(4C) (6A . For exatic mark . This evcarRNAs)  rather tectively . The m6Are-mRNAs , increasre-mRNAs , transcrre-mRNAs , regulatre-mRNAs  developmre-mRNAs . Furtheritiation . Perturb-renewal . Moreoveed mRNAs .5C and its oxidized derivatives across both the genome and the transcriptome. Maintenance of effective, time regulated, and lineage-specific methylation-demethylation dynamics is needed for cellular homeostasis and responses to diverse stimuli. Mutations in epigenetic and epitranscriptomic modifiers can deregulate normal cellular differentiation and programmed growth control. As a consequence of altered patterns of hm5C and m5C, mainly characterised by global hypomethylation and focal hypermethylation, multiple stages of tumorigenesis including initiation, progression and metastasis, are promoted. Moreover, numerous reports correlate the loss of hm5C with poor prognosis. Restoration of proper methylation-demethylation dynamics in cells could be achieve with several approaches. Identification of druggable targets of both main players  and pivotal intermediates (miRNAs or lncRNAs) is crucial for the development of classical inhibitors or RNA-based drugs. A combination of epigenetic therapy and classical chemotherapy is a promising approach aiming at reducing tumour growth and self-renewal characteristics, while reducing chemoresistance (A growing body of evidence suggest that epigenetic and epitranscriptomic dynamics are profoundly interconnected. Methyltransferases (“writers”) and demethylases (“erasers”) functionally cooperate and compete to maintain the appropriate amount of msistance .5C and m6A)   as versa


Nevertheless, the possible participation of CD8+ T cells in TB-IRIS development remains unclear.Tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is a clinical aggravation of TB symptoms observed among a fraction of HIV coinfected patients shortly after the start of antiretroviral therapy (ART). Of note, TB-IRIS is characterized by exacerbated inflammation and tissue damage that occurs in response to the elevated production of CD4+ T cell memory subsets and their association with circulating inflammation-related molecules in TB-HIV coinfected patients initiating ART.We performed a comprehensive assessment of the composition of CD8+ T cells during the onset of IRIS and that the levels of these cells positively correlate with baseline mycobacterial smear grade. TB-IRIS individuals exhibited higher frequencies of effector memory and lower percentages of naïve CD8+ T cells than their Non-IRIS counterparts. In both TB-IRIS and Non-IRIS patients, ART commencement was associated with fewer significant correlations among memory CD8+ T cells and cells from other immune compartments. Networks analysis revealed distinct patterns of correlation between each memory subset with inflammatory cytokines suggesting different dynamics of CD8+ T cell memory subsets reconstitution. TB-IRIS patients displayed lower levels of memory cells positive for CXCR3 (a chemokine receptor that plays a role in trafficking activated CD8+ T cells to the tissues) than Non-IRIS individuals before and after ART. Furthermore, we found that CXCR3+ naïve CD8+ T cells were inversely associated with the risk of TB-IRIS development. On the other hand, we noticed that the frequencies of CXCR3+ effector CD8+ T cells were positively associated with the probability of TB-IRIS development.We found that TB-IRIS individuals display higher frequencies of Antigen-experienced CD8+ T cell subsets reconstitution after ART initiation. Moreover, our data point to a differential association between the frequencies of CXCR3+ CD8+ T cells and the risk of TB-IRIS development. Collectively, our findings lend insights into the potential role of memory CD8+ T cells in TB-IRIS pathophysiology.Our data suggest that TB-IRIS individuals display a distinct profile of memory CD8 Mycobacterium spp infection-related diseases remain as major threats to human health, being associated with substantially high rates of morbidity and mortality worldwide, especially in developing countries (Mycobacterium tuberculosis (Mtb) which produces a spectrum of manifestations including latent infection, subclinical disease, and both active pulmonary and extrapulmonary tuberculosis (TB)  infection considerably tips the balance towards the mitigation of host protective responses (ountries . Noticeasis (TB) . Interesesponses . Of noteesponses .+ T cells results in full activation of previously primed Mtb-harboring macrophages. This ultimately leads to an uncoordinated production of pro-inflammatory molecules and considerable inflammation-driven tissue damage  and ART  may manifest a paradoxical or unmasking worsening of TB symptoms associated with a considerable clinical deterioration in a reaction known as immune reconstitution inflammatory syndrome (IRIS) . Clinicae damage . Identif and ART .+ T cells plays a pivotal role in TB-IRIS immunopathogenesis (++CD16+) monocytes before ART is associated with an increased risk of IRIS development  CD8+ T cells in TB-IRIS patients that arguably could be a compensatory mechanism in the face of severe CD4+ lymphopenia  in TB-IRIS patients. Further dissection of the memory compartment revealed that these patients displayed augmented levels of effector memory cells. Of interest, by devising a Spearman correlation-based approach, we noticed differential patterns of correlation between the CD8+ T cell memory compartment and surrogate markers of cellular activation and inflammation in our study population. Additionally, the magnitude of naïve CD8+ T cell reconstitution after ART was substantially higher among Non-IRIS individuals when contrasted to the TB-IRIS arm of our study. Furthermore, we found that the frequencies of a subpopulation naïve CD8+ T cells expressing CXCR3+ were independently associated with a decreased probability of TB-IRIS development. Our findings provide insights into the potential role of CD8+ T cell memory subset reconstitution in the pathogenesis of TB-IRIS.Here we sought to perform a comprehensive assessment of the memory CD8All clinical investigations were carried out following the principles disclosed in the Declaration of Helsinki. Written informed consent was obtained from all participants before any study procedures. This study was approved by the Scientific Advisory Committee and Institutional Ethics Committee of the National Institute for Research in Tuberculosis (Chennai) and registered on Clinicaltrials.gov (NCT00933790).The present study is a retrospective analysis of clinical, laboratory, and microbiologic data collected from a previously published cohort . The IndClinical evaluation and blood samples were collected at pre-ART (baseline) and at the time of IRIS occurrence or equivalent time point . IRIS was diagnosed by following a modified International Network for the study of HIV-associated IRIS (INSHI) guidelines as previously described . This toConcentrations of C-reactive protein (CRP) , Eotaxin, Fibroblast growth factor (FGF) – basic, FMS-like tyrosine kinase 3 ligand (FLT3L), Granulocyte colony-stimulating factor (G-CSF), intestinal fatty acid binding protein (I-FABP) , Interferon (IFN)-α, IFN-β, IFN-γ, Interleukin (IL)-1Ra, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, Interferon-γ-induced protein 10 (IP-10), Monocyte chemoattractant protein (MCP)-1, Macrophage inflammatory protein (MIP)-1, MIP-1α, MIP-1β, Platelet-derived growth factor (PDGF), Regulated on activation normal T cell expressed and secreted (RANTES), soluble CD14 (sCD14), soluble CD163 (sCD163), soluble GzB, soluble Programmed cell death protein (PD) -1, soluble Tissue factor (sTF), Transforming Growth Factor (TGF)-β, Tumor Necrosis Factor (TNF)-α, and vascular endothelial growth factor (VEGF) were assessed using Luminex assay technology  were assessed in cryopreserved plasma samples maintained at −80°C.+CD45RO+), naïve (CD27+CD45RO−), effector (CD27−CD45RO-), and effector memory , Th2 (CXCR3-CCR6-), and Th17 (CXCR3-CCR6+). Briefly, this characterization was conducted by staining aliquots of 250 µL of whole blood on the day of the participant’s visit with the following antibodies: CXCR3, CD3, CD4, CD8, HLA-DR, PD-1, Ki-67, and GzB. All antibodies were obtained from eBioscience , Biolegend , BD Biosciences , and Life Technologies . The antibody panel was prepared in PBS with 1% BSA for 30 minutes at room temperature. Data were acquired on a BD FACS Canto II flow cytometer (BD Biosciences). All compensation and gate analyses were conducted in FlowJo 9.5.3 .In order to dissect the immunophenotype of T cells, we evaluated markers associated with activation (HLA-DR), exhaustion (PD-1), proliferation (Ki-67), and cytotoxicity (GzB). Phenotypic identification of memory subsets was based on the expression of CD45 and CCR7 . Throughout the text, a p-value of <0.05 was considered statistically significant after adjustments for multiple comparisons (Holm-Bonferroni’s correction method). The statistical analyses were performed using GraphPad Prism 9.0 , the ggplot2 R package (version 3.10), and STATA 9.0 .Median values with IQR or frequencies of variables were compared using the Mann-Whitney + T lymphocyte frequencies in the peripheral blood following treatment initiation  CD8+ T cells in individuals who developed TB-IRIS or not. We tested this by employing a flow cytometric approach that considered the expression of CD45RO and CCR7 to identify the following: naïve cells (CD27+CD45RO−) and Antigen (Ag)-experienced cells . During IRIS onset, TB-IRIS participants displayed higher percentages of memory and lower percentages of naïve CD8+ T cells when contrasted to Non-IRIS individuals  to assess how efficiently memory CD8+ T cell subsets could distinguish our study participants at each timepoint  CD8+ and CD4+ T cells, cytotoxic (GzB+) CD4+ T cells, Th1 and Th2 CD4+ T cells, central memory CD4+ T cells, as well as total and PD-1+ T regulatory (T reg) cells, and effector CD4+ T cells.Before ART, Non-IRIS participants exhibited a higher number of correlations between memory CD8+ T cells displayed fewer correlations with other immune cells. Ag-experienced cells were correlated with central memory and effector memory CD4+ T cells, and PD-1+ Tregs. Conversely, naïve CD8+ T cells from TB-IRIS patients showed a high number of significant correlations. Naïve CD8+ T cells negatively correlated with the total number of CD8+ T cells, GzB+ CD8+ and CD4+ lymphocytes, activated and Ki67+ CD4+ and effector memory CD4+ T cells, while positively correlated with the frequencies of naïve CD4+ T cells.In the TB-IRIS group, pre-ART frequencies of Ag-experienced CD8+ T cells and PD-1+ CD8+, activated CD4+, and Th17 T cells. In this same group, naïve CD8+ T cells were negatively correlated with the total number of CD8+ and GzB+ T lymphocytes as well as central memory CD4+ T cells. Among TB-IRIS individuals, naïve CD8+ T cells displayed several negative associations with total frequency of CD8+ T cells, GzB+ CD8+ T cells, activated and proliferating CD4+ T cells, and effector memory CD4+ T cells. Effector memory CD8+ T cells showed strong positive correlations with the total frequency of CD8+ T cells, Tregs, and activated Tregs. Conversely, we were able to detect negative correlations between these cells and PD-1+ T regs in this same group of patients. In summary, our observations suggest substantial differences in associations among CD8+ T cell memory subsets and markers of cellular activation during immune reconstitution in our study population.After treatment initiation, Non-IRIS individuals exhibited positive correlations between central memory CD8+ T lymphocyte subsets following treatment initiation in our study population. We found that the degree of variation (Δ [the absolute number of cells at timepoint 1 minus the baseline counts]) of naïve CD8+ T cells was higher in Non-IRIS patients compared with TB-IRIS  . Of note, we noticed that the variation of central memory T cells was positively correlated with IFN-α and IP-10, while negatively associated with IL-2, IL-5, and IL-13. Additionally, we found that the Δ of effector memory cells were correlated with sCD163, MIP-1a, and MIP-1b, whereas the variation of effector cells displayed correlations with IFN-α, IL-2, IL-5, IL-13, IP-10, and IFN-β.Concerning TB-IRIS participants, we detected that the Δ of naïve CD8+ T lymphocyte subset reconstitution displays different correlation patterns with inflammatory mediators in TB-IRIS patients, which may have implications for the pathology of TB-IRIS.Collectively, our observations suggest that the magnitude of memory CD8+ T cell memory compartment seen between TB-IRIS and Non-IRIS groups was related to a differential commitment to an effector fate during IRIS onset. To evaluate this possibility, we employed a flow cytometric approach to measuring the expression of CXCR3 + in the memory subsets of CD8+ T cells. Of note, CXCR3 is a chemokine receptor that plays a role in trafficking activated CD8+ T cells to peripheral tissues  ]). On the other hand, the pre-ART percentages of CXCR3+ effector CD8+ T cells were associated with a higher risk of TB-IRIS ]).Next, we applied a multivariate regression model using the pre-ART frequencies of CXCR3+ memory CD8+ T cells at timepoint 1, we noted that increased frequencies of CXCR3+ naïve CD8+ T cells were associated with reduced risk of TB-IRIS development ]). Conversely, we found that higher percentages of CXCR3+ effector CD8+ T cells were independently associated with a higher risk of TB-IRIS ]). Collectively, our data suggest that the abundance of CXCR3+ subpopulations of naïve and memory CD8+ T cells could be employed to assess the risk of TB-IRIS development.Additionally, by applying the same model to the frequencies of CXCR3+ T lymphocyte memory compartment in the face of ART-mediated immune reconstitution in highly immunosuppressed TB-HIV coinfected individuals. Here, we report an inverse association between the frequencies of naïve CXCR3+ CD8+ T cells and the probability of TB-associated IRIS occurrence. Conversely, we also detected that the frequencies of the CXCR3+ effector CD8+ T cell subset were associated with an increased risk of TB-IRIS. There is robust evidence on the contributions of CD8+ T cells in the mobilization of appropriate immune responses in several infectious diseases, including HIV infection and TB . On the other hand, among TB-IRIS the variation in memory T cell subsets exhibited a higher number of correlations with inflammatory mediators. These results seem in good agreement with previous reports of increases of Th1 inflammatory mediators  in TB-IRIS , 24. Onede novo differentiation of naïve T cells in the face of CD4+ T cell rise or to a redistribution of previously differentiated memory CD8+ T cells from lymphoid tissues to the hematogenous compartment. Regarding the quantitative recovery of CD4+ T cells, it has been to occur in two main phases. The first phase, which happens from 1 to up to 6 months after treatment initiation, is characterized by the redistribution of memory T cells that were trapped in lymphoid organs to the periphery, coupled with a substantial reduction in viral load, whereas the other phase can last many years and is characterized by a slow and gradual increase of CD4+ cells , therefore increasing the trafficking of CXCR3+ CD8+ T cells into sites of inflammation. The abundance of CXCR3+ ligands in the settings of TB-IRIS and the identification of cell populations that secrete them are warranted to be conducted.We also wished to ascribe whether the observed heterogeneity in memory compartment reconstitution between our study groups was due to 4+ cells . Along tribution . Our ana+ T cells in the immunopathogenesis of TB-IRIS.The limitations of our study include the small number of patients included in our study cohort with a large number of comparisons and the lack of functional assays to assess cells. Another important limitation is the lack of tissue, to evaluate T-cells in areas of infection and inflammation. The strengths of our study include the selection of a well-described cohort of patients who were not previously treated for either HIV infection or TB, had close clinical follow up along with detailed microbiologic evaluation and same day flow cytometric analysis before the administration of anti-inflammatory medications at the IRIS timepoint. Taken together, our findings provide significant insight into the role of immunological memory of CD8The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Clinicaltrials.gov (NCT00933790). The patients/participants provided their written informed consent to participate in this study.The studies involving human participants were reviewed and approved by Scientific Advisory Committee and Institutional Ethics Committee of the National Institute for Research in Tuberculosis (Chennai) and registered on BA, IS, SS, BP, and AS conceptualized the study. BA and IS supervised the immunological study. GN and SS supervised the clinical study. IS, SS, and AS acquired funding for research. GN, RS, KS, BP, IS, and BA performed the clinical assessments. SA, KN, KA, BA, and LA performed the experiments. RT, BB-D, MA-P, and AQ analyzed the data. RT and BA drafted the first version of the manuscript. All the authors revised the manuscript.This work received support from the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID/NIH) and by the Intramural-to-India grant from the US-India Co-operative research program. This study was also financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Finance Code 001). The work of BA is supported by the Intramural Research Program of the Oswaldo Cruz Foundation (FIOCRUZ) and the National Council for Scientific and Technological Development (CNPq), Brazil. RT, MA-P and BB-D were supported by Ph.D. fellowships from CAPES.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.


Previous studies had shown that lncRNA HULC exhibited different effects in human cancers. However, the role of HULC was not reported in osteosarcoma. Hence, we designed this research to explore the function of HULC in osteosarcoma.  Firstly, HULC expression was measured in osteosarcoma tissues and cells via the RT-qPCR assay. The protein expression was detected through western blot. Then, CCK-8 and Transwell assays were conducted to measure cell proliferation, migration, and invasion.  The expression of HULC was obviously higher in osteosarcoma tissues and cells compared with normal control. Moreover, cell proliferation, migration, and invasion were inhibited by HULC knockdown in osteosarcoma cells. HULC overexpression markedly increased osteosarcoma cell proliferation and tumor size in vivo. Furthermore, HULC increased the activity of AKT-PI3K-mTOR pathway by blocking PTEN in osteosarcoma cells. LY294002 inhibited the phosphorylation of AKT, mTOR, and PI3K. Overexpressing HULC enhanced cell migration and invasion of SAOS-2 cells and MG63 cells, while LY294002 reversed the effects.  HULC enhanced the progression of osteosarcoma through targeting PTEN. Osteosarcoma is caused by bone cell abnormal differentiation and proliferation. Consistent with medicine information, the incidence of osteogenic sarcoma is around 0.2–3/100000 per annum , 5. ThusHigh-throughput transcriptome sequencing analysis has shown that only 2% of the human genome can be transcripted into proteins, and 98% can be transcripted into noncoding RNAs . The funlncRNA HULC was first reported by Panzitt et al. in 2007, which was overexpressed lncRNA in human hepatocellular carcinoma . HULC geTherefore, we investigated the interaction of HULC and PTEN in promoting osteosarcoma cell proliferation and migration.The human osteosarcoma cell lines, including Saos-2, U-2OS, and MG63, were selected as subjects for subsequent analysis. All cells were purchased from the American Type Culture Collection (ATCC) .ΔΔCt− method.Total RNA was extracted by TRIzol reagent . Subsequently, the RNA was reversely transcribed to the cDNA. The M-MLV reverse transcriptase  was used for reverse transcription reaction. SYBR-Green Real-Time Master Mix  was applied for qRT-PCR reaction. Finally, the data were calculated with 2HULC P1: 5′-AACCTCCAGAACTGTGAT-3′ and HULC P2: 5′-CATAATTCAGGGAGAAAG-3′β-Actin P1: 5′-CTTCCTTCCTGGGCATGGAG-3′ and β-actin P2: 5′-GGAACGCTTCACGAATTTGC-3′β-actin . After washing and incubating with the second antibodies, the abundance of the proteins was analyzed by enhanced chemiluminescence (ECL).The cells were treated with RAPA buffer  on ice for 30 min. BCA kit  was applied for the measurement of protein concentration. Subsequently, total proteins were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then were transferred on polyvinylidene fluoride membranes . The membranes were treated with 5% fat-free milk for 2 h and then were incubated with the antibodies of AKT , p-AKT , PI3K , p-PI3K , mTOR , p-mTOR , PTEN , or In short, the specific oligonucleotides targeting HULC were synthesized by Genepharmacy Technology (China): sense: 5′-GATCCGCCACATGAACGCCCAGAGATTTTCAAGAGAAATCTCTGGGCGTTCATGTGGTTTTTTG-3′ and antisense: 5′-AATTCAAAAAACCACATGAACGCCCAGAGATTTCTCTTGAAAATCTCTGGGCGTTCATGTGGCG-3′. After that, the plasmids and control were transfected into Saos-2 and MG63 cells. RT-qPCR was performed to confirm the knockdown efficiency of HULC.4 cells were seeded into 96-well plates and cultured for 1, 2, 3, and 4 d. Subsequently, osteosarcoma cells were incubated with CCK-8 at 37°C for 1 h. The absorbency at 450 nm of the cells was measured with microplate reader Thermo Plate .The cell viability was detected using Cell Counting Kit-8 (CCK-8). Briefly, 1 × 104 cells and 100 μl serum-free medium were injected into upper chamber. For Transwell invasion assay, Matrigel-coated chamber was used for invasion assay, and 3 × 104 cells were plated in 100 μl serum-free medium in the upper. In both assays, 500 μl of the medium and 20% FBS were added to the lower chamber. Subsequently, the cells were cultured at 37°C and 5% CO2 for 24 h. After that, the cells were fixed with 100% methanol for 30 min, and then, the cells in upper chamber were removed. Finally, the cells were stained with 0.5% crystal violet  for 20 min for cell count.For migration assay, 2 × 106 HULC overexpression cells. After 5 week, the weight of the tumors was measured.Male BALB/c nude mice (6 weeks) were obtained from HFK Biosciences and maintained under pathogen-free conditions. The experiment was approved by the Shandong Provincial Hospital Affiliated to Shandong First Medical University. For tumor propagation analysis, the mice accepted the subcutaneous injection including 2 × 10t-tests were applied for difference analysis. P < 0.05 was considered statistically significant.SPSS 20.0  was applied for data analyses. The Kaplan-Meier method and log-rank test were applied for the visualization of survival curves. Moreover, one-way analyses of variance and two-tailed Student's First, the expression of lncRNA HULC abundance was examined in osteosarcoma tissue and normal tissue using RT-qPCR. The expression of HULC in tumor tissues was higher than normal tissues . SimilarThen, the effect of downregulation HULC on osteosarcoma cells was investigated. First, HULC was knockdown in osteosarcoma cell lines successfully Figures . In CCK-Overexpression experiment was performed to detect the effect of HULC in osteosarcoma cell proliferation. In SAOS-2  and MG63In order to investigate the interaction between HULC and PTEN, we overexpressed HULC, and western blot was applied to detect the expression of PTEN and other proteins . We founTo explore whether the effect of HULC on osteosarcoma cells depends on PI3K/Akt/mTOR signaling pathway, we added LY294002, an inhibitor of PI3K signaling pathway. LY294002 inhibited the phosphorylation of AKT, mTOR, and PI3K . OverexpOf all bone cancers, osteosarcoma is the second leading cause of death in children . OsteosaκB signaling pathway that promotes the development of lung squamous cell carcinoma [Previous reports have shown that lncRNA is involved in the formation and development of a variety of tumors , mainly arcinoma . The prePI3 kinase and PTEN are major modulators of the PI3K pathway, regulating cell growth, proliferation, and survival. PTEN regulates PI3K signaling by dephosphorylating the lipid signaling intermediate PIP3 . MutatioIn conclusion, this study supported that the expression of lncRNA HULC was increased in osteosarcoma, which enhanced the progression of osteosarcoma in vivo and in vitro.


E/Z-selective addition of ACPs with imines for the synthesis of dienyl substituted amines has been developed via palladium- and Brønsted acid co-catalysis. A range of synthetically valuable dienyl substituted amines were effectively prepared with good to excellent yields and excellent enantio- and E/Z-selectivities.Transition-metal catalyzed functionalization of ACPs has been widely investigated in cycloaddition and 1,3-difunctionalization reactions. However, the transition metal catalyzed nucleophilic reactions of ACPs have rarely been reported. In this article, an enantio-, site- and  E/Z-selective addition of ACPs with imines for the synthesis of dienyl substituted amines has been developed via palladium- and Brønsted acid co-catalysis.An enantio-, site- and  In addition to their high strain energy, the presence of the double bond, which allows coordination to the transition-metal, leads to an additional variety of activation processes. Their unique structural and electronic properties have therefore attracted considerable interest both in synthetic and mechanistic studies. A series of very interesting and characteristic transformations have been developed in the past decades.4 Among them, the ring-opening and ring-expansion reactions of ACPs are usually favorable owing to the concomitant release of cyclopropane ring strain.Three-membered carbocycles have found many applications not only as versatile building blocks for organic chemistry5 Generally, the reactions of ACPs with transition-metal catalysis can occur via two different reaction patterns 2/BuPAd2 catalyst system in THF -Ph-BPE (L1) gave the desired product in 6% yield and 2% ee . 3aa was obtained in high yield and excellent ee nd 2% ee , entry 1nd 2% ee , entry 2nd 2% ee , entry 3N-Ts imine 2a under the optimized reaction conditions. As summarized in E,E-isomers, and only trace amounts of E,Z-isomers were observed in some cases. The structure of the products was assigned based on X-ray crystallography analysis of 3ba as a representative example.12With the optimized reaction conditions in hand, we turned our attention to explore the substrate scope of this asymmetric 1,4-addition reaction. First, a range of substituted ACPs 1 was applied to react with N-Ts imines 2 possessing different substitutions at different positions of the phenyl ring were successfully applied in this reaction and produced the corresponding products in good to excellent yields with excellent enantioselectivities. In addition, a 2-naphthyl-based imine 2l also reacted smoothly and gave the desired product 3al in a 75% yield and 87% ee. Heteroaryl groups such as 3-thiophenyl were tolerated as well, delivering the corresponding product 3ak in high yields with excellent enantioselectivity. N-Sulfonyl substituted imines 2m and 2n were also suitable substrates in this reaction and produced the corresponding products 3am and 3an, respectively. However, no desired reaction was observed when imines with N-phenyl and N-butyl groups were used in this reaction.Then, we further examined the scope of the imine 2 to demonstrate the generality of this reaction . A group10,11 a plausible catalytic cycle is proposed in in situ generated Pd(0) to the proximal C–C bond of ACPs 1 generates a cyclic Pd(ii) complex I. The cyclic Pd(ii) complex I undergoes β-H elimination and reductive elimination to give an η2-coordinated 1,3-diene intermediate II. Then, intermediate II undergoes enantioselective cyclopalladation with imines 2 to give a cyclic palladium intermediate III. Intermediate III was then protonated by Brønsted acid to give complex IV, which underwent reductive elimination to release the addition product 3 and regenerated Pd(0) for the next catalytic cycle. The use of Brønsted acid activated the imines and provided an extra means to tune the enantioselectivity of the products.On the basis of the experimental results and the previous literature,E/Z-selective addition of ACPs with imines via palladium- and Brønsted acid co-catalysis. A range of synthetically valuable dienyl substituted amines were effectively prepared with good to excellent yields and excellent enantio- and E/Z-selectivities.In summary, we have developed an enantio-, site- and All experimental data and detailed procedures are available in the ESI.†J.-B. P. conceived and directed the project. X.-L. L. performed the experiments. H.-Z. L. and L.-Q. T. participated in substrate synthesis and discussions. X.-L. L. and J.-B. P. wrote the manuscript and ESIThere are no conflicts to declare.SC-014-D2SC05674G-s001SC-014-D2SC05674G-s002


Background: Although immune checkpoint inhibitors have opened a new mode of treatment for solid tumors, their efficacy in nasopharyngeal carcinoma (NPC) needs to be further investigated. Inhibitors of the PD-1/PD-L1 immune checkpoint are one of the hot topics in tumor immunotherapy. Programmed death ligand-2 (PD-L2) is a less studied ligand of PD-1 and has not yet been fully explored, especially in NPC. Understanding the clinical significance of PD-L2 expression, together with immune cell infiltration, might provide clues for biomarker screening in NPC immunotherapy. This study aimed to evaluate the role of PD-L2 as a prognostic factor for NPC patients as well as its role in immune regulation.Methods: Immunohistochemistry (IHC) was performed on a tissue microarray including 557 NPC specimens using PD-L2 antibody. The immune cell markers CD4, FOXP3 and CD68 were also stained and quantified. The expression of PD-L2 exhibited different spatial patterns among NPC tumor and stromal tissues.Results: A total of 90.8% of the cases showed membranous PD-L2 expression in tumors, and 80.8% showed membranous PD-L2 expression in stromal tissue. High stromal expression of PD-L2 predicted favorable overall and disease-free survival of NPC patients and was negatively correlated with tumor size, recurrence or metastasis and clinical stage. In contrast, high tumor abundance of PD-L2 correlated with poor disease-free survival, but had no obvious correlation with clinicopathological parameters. Multivariate analysis indicated that stromal PD-L2 was an independent and favorable prognostic factor. Furthermore, we found a positive correlation between stromal PD-L2 expression and the infiltration of CD68+ macrophages and CD4+Foxp3+ Treg cells in NPC stromal tissues .Conclusions: Our results suggest that different PD-L2 expression patterns have distinct predictive values. PD-L2 expressed on stromal cells might play a role in the regulation of NPC progression, and involve in immune activation in the tissue microenvironment and have an independent good prognosis for NPC patients. NPC is one of the most common head and neck malignancies and is highly prevalent in Southeast Asia, North Africa and southern China Nevertheless, the unique immune environment of EBV-associated NPC indicates the potential benefits of immunotherapy based on immune checkpoint inhibitors (ICIs). Programmed cell death-1 (PD-1) is mainly expressed on activated T cells and acts as an immune activation reaction of tumors after the combination of inhibitory receptor and ligand (PD-L1). In recent years, the application of inhibitors targeting PD-1 and PD-L1 in solid tumors has made substantial progress. In several clinical trials exploring the role of PD-1/PD-L1 inhibitors in NPC, Fang W, Ma B and Hsu C showed a good objective response rate with immune checkpoint inhibitors in patients with recurrent or metastatic NPC PD-1 has two ligands: PD-L1 and programmed death ligand-2 (PD-L2). PD-L1 is the main ligand for PD-1 and is one of the major targets for cancer immunotherapy. Multiple studies are available on the clinical significance of PD-1/PD-L1 in NPC, and the prognostic value remains controversial. Meta-analysis and IHC analysis showed that PD-L1 overexpression in NPC was associated with poor overall survival (OS) + macrophages and CD4+Foxp3+ Treg cells in the tumor microenvironment in NPC.Here, to explore the role of PD-L2 in the NPC tumor microenvironment, we first examined PD-L2 expression in NPC tumor cells and stromal cells using the IHC method. Second, we analyzed the correlation between the PD-L2 expression level and the clinical factors and outcomes of NPC patients. Finally, we evaluated the possible immune-regulatory function of PD-L2 on CD68In this study, 557 NPC specimens were collected at the Department of Pathology, Sun Yat-Sen University Cancer Center (SYSUCC), Guangzhou, China, between January 2001 and December 2003. The inclusion criteria of patients and follow-up were the same as those in previous publications Paraffin-embedded specimens were obtained from a previously constructed tissue microarray (TMA) + antibodies were used to identify T lymphocytes , and FoxP3 antibodies were used to identify regulatory T cells . Immunohistochemistry was performed as double staining using a CD4-specific antibody and a FoxP3-specific antibody. For detection, a Polymer-Kit and Fast Red and Polymer-Kit and Fast brown  were used according to the manufacturer's instructions. The IHC results were evaluated and scored as described previously + macrophages and CD4+Foxp3+ Treg cells in the tumor and stroma were counted within the areas of 6 high-power fields (400X).IHC was performed to examine PD-L2, CD68, CD4 and Foxp3 expression in NPC tissue specimens. Primary antibodies against PD-L2 , CD4 , Foxp3 , and CD68  were used in this study. Formalin-fixed paraffin-embedded tissue specimens were processed in tissue microarrays with cores of 2 mm diameter. CD4Receiver operating characteristic (ROC) curve analysis was performed to determine the cutoff score for a “high expression” designation with the 0,1-criterion implemented in SPSS software 2 test or Fisher's exact probability method was used for comparisons between groups. The survival rate was analyzed by Kaplan-Meier and log-rank methods. The Cox regression model was used for multivariate analysis. Associations between PD-L2 expression and the number of CD68+ macrophages or CD4+Foxp3+ Treg cells were examined by using Pearson correlation and independent t tests. P<0.05 was regarded as statistically significant.Statistical analysis was performed using SPSS . The survival time was calculated monthly, and the OS time was calculated from the date of initial diagnosis to the date of death or the last follow-up. The χAmong the 557 patients, 415 (74.5%) were male, and the median age was 46 years (range 19-78 years). Among these patients, 139 patients (25.0%) were diagnosed with nonkeratinizing differentiated carcinoma (NKDC), and 402 patients (72.2%) were diagnosed with nonkeratinizing undifferentiated carcinoma (NKUC), 16 patients (2.8%) were diagnosed with keratinizing squamous cell carcinoma (KSCC). One hundred forty-seven patients (26.4%) were in stage I and stage II, and 410 patients (73.6%) were in stage III and stage IV. The median follow-up time was 58.63 months (range 2 to 99 months). Of all the patients, 184 (33.0%) died, and 208(37.3%) presented with disease progression during the five-year follow-up period. Detailed clinical information is shown in The expression of PD-L2 protein was determined in 557 NPC tumors from which five-year follow-up information was available by using IHC with the specific antibody. Overexpression of PD-L2 was observed in the membrane of NPC cells with different intensities Figure . A totalROC curve analysis showed that stromal PD-L2 expression has some sort of predictive value in NPC, with the maximum area under the curve (AUC) reaching 0.618 Figure .2-test analysis, we found that high stromal expression of PD-L2 was negatively correlated with age , tumor T classification , recurrence and metastasis (Pearson's R=-0.127 P=0.003) and clinical stage (Pearson's R=-0.111 P=0.009), respectively , but there was no significant association with other clinicopathologic features, such as age, sex, T classification, N classification, recurrence and metastasis and clinical stage  NPC tissues. With χ2 = 10.006, P = 0.002, Figure 2=9.502, P=0.001, Figure The 5-year overall and disease-free survival rates of the cohort of 557 NPC patients were 68.7% and 62.6%, respectively Figure A and 3B.Univariate Cox proportional hazard regression analysis showed that PD-L2 stromal expression (P=0.002), age (P=0.001) and well-known clinicopathologic prognostic parameters, such as T classification (P<0.001), N classification (P=0.001), recurrence or metastasis (P=0.005) and clinical stage (P<0.001), were significantly associated with survival Table . Moreove+ macrophages and CD4+Foxp3+ Treg cells in the tumor and stroma, respectively, which have been implicated in the tumor microenvironment of previous reports + macrophages  and CD4+Foxp3+ Treg cells  with the new type to form a supercomplex, initiates a series of downstream events such as downstream target gene transcription and cytoskeletal recombination, mediates T-cell amplification, and then plays an immune-activating role The differential effects of PD-L2 on tumor cells and stromal cells may be due to different biological roles resulting from the binding of different receptors. Tumor cells may be combined with PD-1 to play an immune escape role and promote tumor progression; however, stromal cells may contribute to an antitumor immune response by associating with the unknown second receptor. Current research on PD-L2 is still in the initial stage, but the development of a study of PD-L2 and its various receptors will help further explore the effect and mechanism of the tumor immune microenvironment in tumor occurrence and development and provide a new set of therapeutic strategies.Supplementary figure and table.Click here for additional data file.


Small nucleolar RNAs (snoRNAs) are a highly expressed class of non-coding RNAs known for their role in guiding post-transcriptional modifications of ribosomal RNAs and small nuclear RNAs. Emerging studies suggest that snoRNAs are also implicated in regulating other vital cellular processes, such as pre-mRNA splicing and 3′-processing of mRNAs, and in the development of cancer and viral infections. There is an emerging body of evidence for specific snoRNA’s involvement in the optimal replication of RNA viruses. In order to investigate the expression pattern of snoRNAs during influenza A viral infection, we performed RNA sequencing analysis of the A549 human cell line infected by influenza virus A/Puerto Rico/8/1934 (H1N1). We identified 66 that were upregulated and 55 that were downregulated in response to influenza A virus infection. The increased expression of most C/D-box snoRNAs was associated with elevated levels of 5’- and 3’-short RNAs derived from this snoRNA. Analysis of the poly(A)+ RNA sequencing data indicated that most of the differentially expressed snoRNAs synthesis was not correlated with the corresponding host genes expression. Furthermore, influenza A viral infection led to an imbalance in the expression of genes responsible for C/D small nucleolar ribonucleoprotein particles’ biogenesis. In summary, our results indicate that the expression pattern of snoRNAs in A549 cells is significantly altered during influenza A viral infection. Influenza viruses are enveloped single-stranded segmented negative-sense RNA viruses of the Orthomyxoviridae family, which are classified into four genera: influenza virus A–D  . The greSmall nucleolar RNAs are a class of small ncRNA 60–300 nt long that are widely present in eukaryotic cells’ nucleolus. In the human genome, snoRNAs are predominantly encoded by intronic regions of both non-coding and protein-coding genes . It is gHowever, recent studies have indicated that some snoRNAs are expressed at different levels with regard to the cell type and biological and environmental factors ,10,11. SMoreover, the accumulated knowledge indicates that the expression of snoRNAs and sdRNAs is often changed in pathological conditions such as cancer or viral infection ,23,24,25Here, we performed small RNA sequencing analysis of influenza-A-virus-infected A549 cells, which revealed that 121 snoRNAs show a significant change in their expression in response to infection. In addition, we showed an association between upregulated expression of most C/D-box snoRNAs and an increase in the level of 5′- and 3′-short RNA derived from these snoRNAs. Based on the parallel analysis of poly (A)+ RNA sequencing data, we examined the correlation between the levels of intronic C/D-box snoRNA and corresponding host genes and the change in the expression of genes responsible for C/D snoRNP biogenesis during influenza A viral infection.For systematic investigation of small nucleolar RNAs’ regulation during influenza A viral infection, we performed RNA sequencing analysis of A/Puerto Rico/8/1934-virus-infected A549 cells. We generated six small RNA and six poly(A) + RNA libraries using two biological replicates of each time point: before infection (0 h) and after infection (24 and 48 h). The sequencing reactions yielded approximately 6.4 and 11.5 million raw reads for the small RNA and poly(A)+ RNA libraries, respectively . Raw reaDifferential analysis of the small RNA expression revealed that large numbers of snoRNAs were upregulated or downregulated in response to influenza A virus infection . An incr6 reads to C/D-box vs. 7.73 × 10³ reads to H/ACA-box for each sample). Secondly, the selected sequencing protocol (75-nucleotide single-end reads) is more suitable for the analysis of the C/D-box snoRNA distribution (an average length is 60–90 nucleotides) then for the analysis of H/ACA-box snoRNAs (an average length is about 150 nucleotides). Thirdly, we focused on the C/D-box snoRNAs as more structure and function diversity has, up to now, been identified among this subclass compared with H/ACA-box [C/D-box snoRNAs were examined in more detail. Firstly, 99% of all reads mapped to snoRNAs were aligned to C/D-box snoRNAs  . Visual To validate our sequencing data, we tested the expression of upregulated SNORD93, SNORD11, SNORD1B, SNORD8, and derived sdRNAs in independent samples of A549 cells infected with influenza A/Puerto Rico/8/1934 virus by real-time quantitative reverse transcription PCR (RT-PCR) with custom stem-loop primers . The RT-In the human genome, most C/D-box snoRNA genes are located within the introns of host genes encoding mRNA or long noncoding RNA A. The biMIG8, RPL7A, SNHG1, SNHG14, SNHG32) contain both upregulated and downregulated snoRNAs located in different introns. Recent studies have shown that the expression of individual snoRNAs and their cognate spliced RNA can be uncoupled via alternative splicing and nonsense-mediated decay [According to our data, the synthesis of most differentially expressed snoRNAs is not correlated with the expression of the host genes. Only 25% of all differentially expressed C/D-box snoRNAs (18 upregulated and 2 downregulated snoRNAs) and corresponding host genes were characterized by significant unidirectional changes 48 h after influenza A virus infection B. To noted decay .The differential expression analysis of genes responsible for C/D snoRNP biogenesis revealed an imbalance in the mRNA level of genes involved in the major steps of snoRNA maturation during influenza A viral infection . While tThe main function of C/D-box snoRNAs is to guide site-specific 2′-O-methylation of rRNA nucleotides . The rRNUsing the method based on the termination of reverse transcription, we analyzed the changes in the target nucleotides of 2′-O-methylation for some differentially expressed snoRNAs . All selected nucleotides were characterized by a trend toward decreased 2′-O-methylation after 48 h of incubation after influenza A virus infection .Influenza A virus (IAV), causing annual seasonal epidemics and global pandemics, is of high public health concern. During the process of IAV infection, disturbances are observed at the different levels of gene expression, including activation of antiviral gene transcription, regulation of alternative splicing, changes in post-transcriptional modifications, and control of the translational machinery ,44,45,46In this paper, through RNA sequencing analysis of A549 human cells infected with influenza A/Puerto Rico/8/1934 (H1N1) virus, we found that a large number of snoRNAs are differentially expressed in human cells during influenza infection. Further, 48 h after infection, 38 and 43 C/D-box snoRNAs and 29 and 13 H/ACA-box snoRNAs were upregulated and downregulated, respectively. The main function of snoRNAs is known to guide site-specific rRNA modifications required for ribosome biogenesis . Recent At present, it is known that the majority of ncRNA expression is dysregulated in response to influenza virus infection ,6. HowevVisual and size distribution analyses of the mapped reads revealed that upregulated C/D-box snoRNAs were associated with the increase in the level of 5′- and 3′-short RNA derived from snoRNAs. Such fragments of snoRNA processing were discovered in the late 2000s and were named sno-derived RNAs ,17. NextPIPOX gene through miRNA-like silencing, contributing to the malignant phenotype of breast cancer [TAF9B gene [Despite the fact that most RNA fragments derived from C/D-box snoRNAs are different in size from miRNAs (17–19 nt or > 27 nt for snoRNAs vs. 21–22 nt for miRNAs) and are not efficiently incorporated into the Ago2 protein, a small number of sno-RNA-derived RNAs may carry out miRNA-like functions ,52. sdRNt cancer . sdRNA-2F9B gene . AccordiIt is worth noting that both previous studies and our study indicate that non-infected cells also contain similar fragments of snoRNA processing. Thus, influenza A virus infection merely influences the activity of the sdRNA formation process. It is known that viruses induce host shutoff when the cells begin to produce viral proteins at the expense of host proteins. The main cause for host shutoff during influenza A virus infection is selective degradation of host mRNAs by PA-X endonuclease. The analysis of the RNA-sequencing and ribosome profiling of A549 cells infected with A/Puerto Rico/8/1934 (H1N1) virus revealed a relative reduction in the RNA (2-4-fold) and translation (2-fold) levels of the main genes responsible for C/D-box snoRNA biogenesis: FBL, NOP56, NOP58, and NHP2L1 (SNU13), 12 h after infection . In our In conclusion, we demonstrated the expression pattern of snoRNAs in A549 human cells infected with influenza A virus. We also showed that some C/D-box snoRNAs are characterized by an increase in the sno-derived RNA level. The regulation of snoRNA biogenesis and processing during infection is yet another example of the intricate and poorly understood interaction between the host and virus. Further study of the interaction between snoRNAs and influenza viruses will likely uncover new functions of snoRNAs and possibly unveil new anti-viral strategies.A/Puerto Rico/8/1934 (H1N1) influenza virus from the collection of Smorodintsev Research Institute of Influenza  was used. A/Puerto Rico/8/1934 influenza virus were propagated in 10-day-old ECE. Virus titers were determined by estimating the 50% tissue culture infectious dose (TCID50) in MDCK cell culture . Virus stocks were stored at −80 °C until use.2.A549 cells  were maintained in DMEM/F12  supplemented with GlutaMAX  and 10% FBS  at 37 °C, 5% CO2. As absorption passed, the inoculum was removed, and fresh media was added. Infected cells were incubated at 37 °C for 24 or 48 h, 5% CO2. At 24 and 48 h after infection, all the culture media were collected, and the titers were measured by the TCID50 assay. The growth kinetics of the influenza A/Puerto Rico/8/1934 (H1N1) virus in the cell lines are presented in Cells were grown in T25 cell culture flasks  until 90–100% monolayer. Influenza A/Puerto Rico/8/1934 virus was diluted in culture media , 1 μg/mL TPCK-trypsin ) to reach a multiplicity of infection of 1 TCID50/cell. Cells monolayers were washed twice by DPBS  and subsequently virus inoculum was added (1 mL/flask) and incubated at 37 °C for 1 h, 5% COTotal RNA and small RNA (< 200 nucleotide length) fractions were extracted from cells and were purified using the phenol-chloroform extraction method followed by isolation on absorption columns using the LRU-100–50 kit  or analogical mirVana™ miRNA isolation kit  and diluted with nuclease-free water . The RNA concentration was assessed using a Qubit RNA HS Assay Kit  with a Qubit 2 fluorometer . The quality of total RNA, expressed as the RNA integrity number (RIN), was determined with a Bioanalyzer 2100 instrument  using an Agilent RNA Pico 6000 Kit  . A thresA total of 12 cDNA libraries + RNA) were prepared from 2 biological replicates of each time point: before infection (0 h) and after infection (24 and 48 h). The construction of cDNA libraries was performed according to a standard protocol using a NEBNext Multiplex Small RNA Library Prep Kit for Illumina  for small RNA fraction, NEBNext Ultra II Directional RNA library preparation kit for Illumina , and NEBNext mRNA Magnetic Isolation Module  for poly(A)+ RNA fraction. For the prepared sequencing libraries, the fragment size distribution was analyzed using a Bioanalyzer 2100 instrument  with an Agilent High Sensitivity DNA Kit  and quantification by a Qubit DNA HS Assay Kit  with a Qubit 2 fluorometer . Libraries were sequenced on an Illumina NextSeq 500 instrument  in 75-base-pair-single-end mode ). Binary Base Call files provided by the Illumina Real-Time Analysis (RTA) software were de-multiplexed and converted into FASTQ format by bcl2fastq2 Conversion Software . The construction of cDNA libraries and massive parallel sequencing were conducted at the Institute of Fundamental Medicine and Biology, Kazan Federal University .p-value < 0.05 and the absolute value of a log2 (FC) > 0.58 [https://www.ebi.ac.uk/biostudies/arrayexpress, accessed on 1 August 2022) under the accession number E-MTAB-12088  and E-MTAB-9511 (poly(A)+ RNA fraction) [The raw data were saved as FASTQ format files. The quality control of the raw and trimmed reads was performed using FastQC  and MultiQC  ,58. Trim) > 0.58 . To dete) > 0.58 ,65; read) > 0.58 ; the obtraction) .RT-qPCR reaction was performed with BioMaster RT-qPCR SYBR Blue , and two-step stem-loop RT-qPCR was performed with Reverse Transcriptase M-MuLV–RH  and BioMaster UDG HS-qPCR  using LightCycler 96 System . The primers employed in this study are presented in −∆∆Ct method, where ∆∆Ct = ∆Ct3.0mM − ∆Ct1.0mM; ∆Ct = CtF/R − CtIn/R. The relative change in the modification level of the target nucleotide was evaluated based on the difference between the obtained values for the study (A549 cells 24 and 48 h after influenza A virus infection) and control (non-infected intact A549 cells) samples. This approach is based on the method of identification of the 2′-O-methylation groups in rRNA by RT-qPCR first presented by Belin et al. [Reverse transcription followed by PCR with modification-specific primers was performed using total RNA samples. The primers employed for analysis of the methylation status of 28S:Am3809 (SNORD79); 28S:Gm4198 (SNORD58a); 18S:Am576 (SNORD93); and 28S:Gm4362 (SNORD1b) are presented in n et al. ,71.