Therapeutic potential activity of quercetin complexes against Streptococcus pneumoniae.

This study investigates quercetin complexes as potential synergistic agents against the important respiratory pathogen Streptococcus pneumoniae. Six quercetin complexes (QCX1-6) were synthesized by reacting quercetin with various metal salts and boronic acids and characterized using FTIR spectroscopy. Their antibacterial activity alone and in synergism with antibiotics was evaluated against S. pneumoniae ATCC 49619 using disc diffusion screening, broth microdilution MIC determination, and checkerboard assays. Complexes QCX-3 and QCX-4 demonstrated synergy when combined with levofloxacin via fractional inhibitory concentration indices ≤ 0.5 as confirmed by time-kill kinetics. Molecular docking elucidated interactions of these combinations with virulence enzymes sortase A and sialidase. A biofilm inhibition assay found the synergistic combinations more potently reduced biofilm formation versus monotherapy. Additionally, gene-gene interaction networks, biological activity predictions and in-silico toxicity profiling provided insights into potential mechanisms of action and safety.

Development and characterization of an antibody that recognizes influenza virus N1 neuraminidases.

Influenza A viruses (IAVs) continue to pose a huge threat to public health, and their prevention and treatment remain major international issues. Neuraminidase (NA) is the second most abundant surface glycoprotein on influenza viruses, and antibodies to NA have been shown to be effective against influenza infection. In this study, we generated a monoclonal antibody (mAb), named FNA1, directed toward N1 NAs. FNA1 reacted with H1N1 and H5N1 NA, but failed to react with the NA proteins of H3N2 and H7N9. In vitro, FNA1 displayed potent antiviral activity that mediated both NA inhibition (NI) and blocking of pseudovirus release. Moreover, residues 219, 254, 358, and 388 in the NA protein were critical for FNA1 binding to H1N1 NA. However, further validation is necessary to confirm whether FNA1 mAb is indeed a good inhibitor against NA for application against H1N1 and H5N1 viruses.

Binding of Akkermansia muciniphila to mucin is O-glycan specific.

The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O-glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O-glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galß1-4GlcNAcß1-R) disaccharide selectively on core2 and core3 O-glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium.

Discovery andsynthesis of novel benzoylhydrazone neuraminidase inhibitors.

Neuraminidase (NA) serves as a promising target for the exploration and development of anti-influenza drugs. In this work, lead compound 5 was discovered through pharmacophore-based virtual screening and molecular dynamics simulation, and 14 new compounds were obtained by modifying the lead compound 5 based on pharmacophore features. The biological activity test shows that 5n (IC50 = 0.13 µM) has a better inhibitory effect on wild-type NA (H5N1), while 5i (IC50 = 0.44 µM) has a prominent inhibitory effect on mutant NA (H5N1-H274Y), both of them are better than the positive control oseltamivir carboxylate (OSC). The analysis of docking results indicate that the good activities of compounds 5n and 5i may be attributed to the thiophene ring in 5n can stretch into the 150-cavity of NA, whereas the thiophene moiety in 5i can extend to the 430-cavity of NA. The findings of this study may be helpful for the discovery of new NA inhibitors.

From genomic insights to clinical hope: Targeting NEU1 in IgA nephropathy.

BACKGROUND: IgA Nephropathy (IgAN), the primary form of glomerulonephritis, presents significant clinical challenges due to its obscure pathogenesis and lack of targeted treatments. We conducted a proteome-wide Mendelian randomization (MR) study to identify therapeutic targets for IgAN. METHODS: Utilizing a plasma proteome dataset comprising 4907 blood plasma proteins as the exposure variable, and renal biopsy-confirmed IgAN cases as the outcome, this study employed MR to pinpoint proteins potentially pathogenic to IgAN. The robustness of our findings was affirmed through external dataset validation, reverse causation testing, and Bayesian colocalization analysis. Additionally, we conducted phenotypic scanning and analyzed downstream metabolites to investigate candidate proteins's biological function. RESULTS: In our study, a significant association was identified between an increase in neuraminidase 1 (NEU1) expression and the risk of IgAN. Specifically, a one standard deviation increase in NEU1 expression was associated with an odds ratio of 11.80 for the development of IgAN (95% confidence interval: 4.03-34.54). This association was substantiated across various statistical models and external validations. Colocalization analysis indicated a shared causal variant between NEU1 expression and IgAN. Furthermore, an increased influenza risk associated with NEU1 was observed, supporting the therapeutic potential of NEU1 inhibitors for IgAN. However, our study found no significant role for neuraminic acid-related metabolites in IgAN's development, suggesting an independent pathway for NEU1's influence. CONCLUSION: This study identifies NEU1 as a promising therapeutic target for IgAN, backed by robust genetic evidence. Future research should explore NEU1's therapeutic potential in diverse populations and clinical scenarios, further establishing its role in IgAN.

Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Design, synthesis, docking, and antiviral evaluation of some novel pyrimidinone-based α-aminophosphonates as potent H1N1 and HCoV-229E inhibitors.

Dialkyl/aryl aminophosphonates, 3a-g and 4a-e were synthesized using the LiClO4 catalyzed Kabachnic Fields-type reaction straightforwardly and efficiently. The synthesized phosphonates structures were characterized using elemental analyses, FT-IR, 1H NMR, 13C NMR, and MS spectroscopy. The new compounds were subjected to in-silico molecular docking simulations to evaluate their potential inhibition against Influenza A Neuraminidase and RNA-dependent RNA polymerase of human coronavirus 229E. Subsequently, the compounds were further tested in vitro using a cytopathic inhibition assay to assess their antiviral activity against both human Influenza (H1N1) and human coronavirus (HCoV-229E). Diphenyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (furan-2-yl) methyl) phosphonate (3f) and diethyl ((2-(5-cyano-6-oxo-4-phenyl-1,6-dihydropyrimidin-2-yl) hydrazinyl) (1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl) methyl) phosphonate (4e) were demonstrated direct inhibition activity against Influenza A Neuraminidase and RNA-dependent RNA polymerase. This was supported by their highly favorable binding energies in-silico, with top-ranked values of -12.5 kcal/mol and -14.2 kcal/mol for compound (3f), and -13.5 kcal/mol and -9.89 kcal/mol for compound (4e). Moreover, they also displayed notable antiviral efficacy in vitro against both viruses. These compounds demonstrated significant antiviral activity, as evidenced by selectivity indices (SI) of 101.7 and 51.8, respectively against H1N1, and 24.5 and 5.1 against HCoV-229E, respectively.

Novel Anti-Viral Properties of the Herbal Extract of Davallia mariesii against Influenza A Virus.

Gu-Sui-Bu, the dried rhizome of Davallia mariesii, is a traditional Chinese herbal remedy with a significant history of treating osteoporosis and inflammatory conditions. However, its potential as an anti-influenza agent and its underlying mechanisms of action remain unexplored. To obtain a more potent extract from D. mariesii and gain insights into its mechanism of action against influenza A virus (IAV), we utilized a partitioning process involving organic solvents and water, resulting in the isolation of butanolic subfractions of the D. mariesii extract (DMBE). DMBE exhibited a broad anti-viral spectrum, effectively inhibiting IAV, with an EC50 of 24.32 ± 6.19 µg/mL and a selectivity index of 6.05. We subsequently conducted a series of in vitro assays to evaluate the antiviral effects of DMBE and to uncover its mechanisms of action. DMBE was found to inhibit IAV during the early stages of infection by hindering the attachment of the virus onto and its penetration into host cells. Importantly, DMBE was observed to hinder IAV-mediated cell-cell fusion. It also inhibited neuraminidase activity, plaque size, and the expression levels of phospho-AKT. In summary, this study provides evidence for the effectiveness of D. mariesii as a complementary and alternative herbal remedy against IAV. Specifically, our data highlight DMBE's capabilities in inhibiting viral entry and the release of virions.

Sialic acids as attachment factors in mosquitoes mediating Japanese encephalitis virus infection.

The role of Culex mosquitoes in the transmission of Japanese encephalitis virus (JEV) is crucial, yet the mechanisms of JEV infection in these vectors remain unclear. Previous research has indicated that various host factors participate in JEV infection. Herein, we present evidence that mosquito sialic acids enhance JEV infection both in vivo and in vitro. By treating mosquitoes and C6/36 cells with neuraminidase or lectin, the function of sialic acids is effectively blocked, resulting in significant inhibition of JEV infection. Furthermore, knockdown of the sialic acid biosynthesis genes in Culex mosquitoes also leads to a reduction in JEV infection. Moreover, our research revealed that sialic acids play a role in the attachment of JEV to mosquito cells, but not in its internalization. To further explore the mechanisms underlying the promotion of JEV attachment by sialic acids, we conducted immunoprecipitation experiments to confirm the direct binding of sialic acids to the last α-helix in JEV envelope protein domain III. Overall, our study contributes to a molecular comprehension of the interaction between mosquitoes and JEV and offers potential strategies for preventing the dissemination of flavivirus in natural environments.IMPORTANCEIn this study, we aimed to investigate the impact of glycoconjugate sialic acids on mosquito infection with Japanese encephalitis virus (JEV). Our findings demonstrate that sialic acids play a crucial role in enhancing JEV infection by facilitating the attachment of the virus to the cell membrane. Furthermore, our investigation revealed that sialic acids directly bind to the final α-helix in the JEV envelope protein domain III, thereby accelerating virus adsorption. Collectively, our results highlight the significance of mosquito sialic acids in JEV infection within vectors, contributing to a better understanding of the interaction between mosquitoes and JEV.

Functional Selectivity of Cannabinoid Type 1 G Protein-Coupled Receptor Agonists in Transactivating Glycosylated Receptors on Cancer Cells to Induce Epithelial-Mesenchymal Transition Metastatic Phenotype.

Understanding the role of biased G protein-coupled receptor (GPCR) agonism in receptor signaling may provide novel insights into the opposing effects mediated by cannabinoids, particularly in cancer and cancer metastasis. GPCRs can have more than one active state, a phenomenon called either 'biased agonism', 'functional selectivity', or 'ligand-directed signaling'. However, there are increasing arrays of cannabinoid allosteric ligands with different degrees of modulation, called 'biased modulation', that can vary dramatically in a probe- and pathway-specific manner, not from simple differences in orthosteric ligand efficacy or stimulus-response coupling. Here, emerging evidence proposes the involvement of CB1 GPCRs in a novel biased GPCR signaling paradigm involving the crosstalk between neuraminidase-1 (Neu-1) and matrix metalloproteinase-9 (MMP-9) in the activation of glycosylated receptors through the modification of the receptor glycosylation state. The study findings highlighted the role of CB1 agonists AM-404, Aravnil, and Olvanil in significantly inducing Neu-1 sialidase activity in a dose-dependent fashion in RAW-Blue, PANC-1, and SW-620 cells. This approach was further substantiated by findings that the neuromedin B receptor inhibitor, BIM-23127, MMP-9 inhibitor, MMP9i, and Neu-1 inhibitor, oseltamivir phosphate, could specifically block CB1 agonist-induced Neu-1 sialidase activity. Additionally, we found that CB1 receptors exist in a multimeric receptor complex with Neu-1 in naïve, unstimulated RAW-Blue, PANC-1, and SW-620 cells. This complex implies a molecular link that regulates the interaction and signaling mechanism among these molecules present on the cell surface. Moreover, the study results demonstrate that CB1 agonists induce NFκB-dependent secretory alkaline phosphatase (SEAP) activity in influencing the expression of epithelial-mesenchymal markers, E-cadherin, and vimentin in SW-620 cells, albeit the impact on E-cadherin expression is less pronounced compared to vimentin. In essence, this innovative research begins to elucidate an entirely new molecular mechanism involving a GPCR signaling paradigm in which cannabinoids, as epigenetic stimuli, may traverse to influence gene expression and contribute to cancer and cancer metastasis.

Bacterial-derived sialidases inhibit porcine rotavirus OSU replication by interfering with the early steps of infection.

Rotavirus infections in suckling and weaning piglets cause severe dehydration and death, resulting in significant economic losses in the pig breeding industry. With the continuous emergence of porcine rotavirus (PoRV) variants and poor vaccine cross-protection among various genotypes, there is an urgent need to develop alternative strategies such as seeking effective antiviral products from nature, microbial metabolites and virus-host protein interaction. Sialidases play a crucial role in various physiopathological processes and offer a promising target for developing antivirus drugs. However, the effect of bacterial-derived sialidases on the infection of PoRVs remains largely unknown. Herein, we investigated the impact of bacterial-derived sialidases (sialidase Cp and Vc) on PoRV strain OSU(Group A) infection, using differentiated epithelial monkey kidney cells (MA104) as a model. Our results indicated that the pretreatment of MA104 with exogenous sialidases effectively suppressed PoRV OSU in a concentration-dependent manner. Notably, even at a concentration of 0.01 µU/mL, sialidases significantly inhibited the virus (MOI = 0.01). Meanwhile, we found that sialidase Vc pretreatment sharply reduced the binding rate of PoRV OSU. Last, we demonstrated that PoRV OSU might recognize α-2,3-linked sialic acid as the primary attachment factor in MA104. Our findings provide new insights into the underlying mechanism of PoRV OSU infections, shedding lights on the development of alternative antivirus approaches based on bacteria-virus interaction.

Surface-Controlled Sialoside-Based Biosensing of Viral and Bacterial Neuraminidases.

Neuraminidases (NA) are sialic acid-cleaving enzymes that are used by both bacteria and viruses. These enzymes have sialoside structure-related binding and cleaving preferences. Differentiating between these enzymes requires using a large array of hard-to-access sialosides. In this work, we used electrochemical impedimetric biosensing to differentiate among several pathogene-related NAs. We used a limited set of sialosides and tailored the surface properties. Various sialosides were grafted on two different surfaces with unique properties. Electrografting on glassy carbon electrodes provided low-density sialoside-functionalized surfaces with a hydrophobic submonolayer. A two-step assembly on gold electrodes provided a denser sialoside layer on a negatively charged submonolayer. The synthesis of each sialoside required dozens of laborious steps. Utilizing the unique protein-electrode interaction modes resulted in richer biodata without increasing the synthetic load. These principles allowed for profiling NAs and determining the efficacy of various antiviral inhibitors.

Site-specific immobilization of the endosialidase reveals QSOX2 is a novel polysialylated protein.

Polysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous, immune, and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases, including cancers and mental health disorders. However, the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase, endoneuraminidase NF (EndoN), and the catalytically inactive polySia lectin EndoNDM, have been extensively used for studying polySia. However, EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species, the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples, while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM, which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein, QSOX2, which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology.

Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

HDAC1-Mediated Downregulation of NEU1 Exacerbates the Aggressiveness of Cervical Cancer.

HDAC1 functions as an oncogene in multi-type cancers. This study aimed to investigate the roles of histone deacetylase 1 (HDAC1) in cervical cancer (CC). mRNA expression was determined using reverse transcription quantitative polymerase chain reaction. The protein-protein complexes was analyzed using co-immunoprecipitation assay. The binding sites between NRF2 and NEU1 were confirmed by chromatin immunoprecipitation assay. Cell viability was detected by CCK-8. Cell proliferation was measured using CCK-8 and colony formation assays. Cell migrative and invasive ability were determined using transwell assay. We found that HDAC1 was upregulated in CC patients and cells. Trichostatin A (TSA) treatment decreased the number of colonies and migrated and invaded cells. Moreover, HDAC1 interacted with NRF2 to downregulate NEU1 expression. NEU1 knockdown attenuated the effects of TSA and enhanced the aggressiveness of CC cells. In conclusion, HDAC1 functions as an oncogene in CC. Targeting HDAC1 may be an alternative strategy for CC.

Pneumococcal sialidase promotes bacterial survival by fine-tuning of pneumolysin-mediated membrane disruption.

Pneumolysin (Ply) is an indispensable cholesterol-dependent cytolysin for pneumococcal infection. Although Ply-induced disruption of pneumococci-containing endosomal vesicles is a prerequisite for the evasion of endolysosomal bacterial clearance, its potent activity can be a double-edged sword, having a detrimental effect on bacterial survivability by inducing severe endosomal disruption, bactericidal autophagy, and scaffold epithelial cell death. Thus, Ply activity must be maintained at optimal levels. We develop a highly sensitive assay to monitor endosomal disruption using NanoBiT-Nanobody, which shows that the pneumococcal sialidase NanA can fine-tune Ply activity by trimming sialic acid from cell-membrane-bound glycans. In addition, oseltamivir, an influenza A virus sialidase inhibitor, promotes Ply-induced endosomal disruption and cytotoxicity by inhibiting NanA activity in vitro and greater tissue damage and bacterial clearance in vivo. Our findings provide a foundation for innovative therapeutic strategies for severe pneumococcal infections by exploiting the duality of Ply activity.

Gene therapy corrects the neurological deficits of mice with sialidosis.

Patients with sialidosis (mucolipidosis type I) type I typically present with myoclonus, seizures, ataxia, cherry-red spots, and blindness because of mutations in the neuraminidase 1 (NEU1) gene. Currently, there is no treatment for sialidosis. In this study, we developed an adeno-associated virus (AAV)-mediated gene therapy for a Neu1 knockout (Neu1-/-) mouse model of sialidosis. The vector, AAV9-P3-NP, included the human NEU1 promoter, NEU1 cDNA, IRES, and CTSA cDNA. Untreated Neu1-/- mice showed astrogliosis and microglial LAMP1 accumulation in the nervous system, including brain, spinal cord, and dorsal root ganglion, together with impaired motor function. Coexpression of NEU1 and protective protein/cathepsin A (PPCA) in neonatal Neu1-/- mice by intracerebroventricular injection, and less effective by facial vein injection, decreased astrogliosis and LAMP1 accumulation in the nervous system and improved rotarod performance of the treated mice. Facial vein injection also improved the grip strength and survival of Neu1-/- mice. Therefore, cerebrospinal fluid delivery of AAV9-P3-NP, which corrects the neurological deficits of mice with sialidosis, could be a suitable treatment for patients with sialidosis type I. After intracerebroventricular or facial vein injection of AAV vectors, NEU1 and PPCA are expressed together. PPCA-protected NEU1 is then sent to lysosomes, where ß-Gal binds to this complex to form a multienzyme complex in order to execute its function.

A pleiotropic role of sialidase in the pathogenicity of Porphyromonas gingivalis.

As one of the keystone pathogens of periodontitis, the oral bacterium Porphyromonas gingivalis produces an array of virulence factors, including a recently identified sialidase (PG0352). Our previous report involving loss-of-function studies indicated that PG0352 plays an important role in the pathophysiology of P. gingivalis. However, this report had not been corroborated by gain-of-function studies or substantiated in different P. gingivalis strains. To fill these gaps, herein we first confirm the role of PG0352 in cell surface structures (e.g., capsule) and serum resistance using P. gingivalis W83 strain through genetic complementation and then recapitulate these studies using P. gingivalis ATCC33277 strain. We further investigate the role of PG0352 and its counterpart (PGN1608) in ATCC33277 in cell growth, biofilm formation, neutrophil killing, cell invasion, and P. gingivalis-induced inflammation. Our results indicate that PG0352 and PGN1608 are implicated in P. gingivalis cell surface structures, hydrophobicity, biofilm formation, resistance to complement and neutrophil killing, and host immune responses. Possible molecular mechanisms involved are also discussed. In summary, this report underscores the importance of sialidases in the pathophysiology of P. gingivalis and opens an avenue to elucidate their underlying molecular mechanisms.

EPO promotes the progression of rheumatoid arthritis by inducing desialylation via increasing the expression of neuraminidase 3.

OBJECTIVE: Erythropoietin (EPO) known as an erythrocyte-stimulating factor is increased in patients with rheumatoid arthritis (RA). Nevertheless, the function of EPO in the process of RA and relative mechanism needs to be further clarified. METHODS: The level of EPO in serum and synovial fluid from patients with RA and healthy controls was determined by . Collagen-induced arthritis (CIA) mice were constructed to confirm the role of EPO on RA pathogenesis. Differentially expressed genes (DEGs) of EPO-treated fibroblast-like synoviocyte (FLS) were screened by transcriptome sequencing. The transcription factor of neuraminidase 3 (NEU3) of DEGs was verified by double luciferase reporting experiment, DNA pulldown, electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR (qPCR) assay. RESULTS: The overexpression of EPO was confirmed in patients with RA, which was positively associated with Disease Activity Score 28-joint count. Additionally, EPO intervention could significantly aggravate the joint destruction in CIA models. The upregulation of NEU3 was screened and verified by transcriptome sequencing and qPCR in EPO-treated FLS, and signal transducer and activator of transcription 5 was screened and verified to be the specific transcription factor of NEU3. EPO upregulates NEU3 expression via activating the Janus kinase 2 (JAK2)-STAT5 signalling pathway through its receptor EPOR, thereby to promote the desialylation through enhancing the migration and invasion ability of FLS, which is verified by JAK2 inhibitor and NEU3 inhibitor. CONCLUSION: EPO, as a proinflammatory factor, accelerates the process of RA through transcriptional upregulation of the expression of NEU3 by JAK2/STAT5 pathway.

The artificial amino acid change in the sialic acid-binding domain of the hemagglutinin neuraminidase of newcastle disease virus increases its specificity to HCT 116 colorectal cancer cells and tumor suppression effect.

BACKGROUND: Oncolytic viruses are being studied and developed as novel cancer treatments. Using directed evolution technology, structural modification of the viral surface protein increases the specificity of the oncolytic virus for a particular cancer cell. Newcastle disease virus (NDV) does not show specificity for certain types of cancer cells during infection; therefore, it has low cancer cell specificity. Hemagglutinin is an NDV receptor-binding protein on the cell surface that determines host cell tropism. NDV selectivity for specific cancer cells can be increased by artificial amino acid changes in hemagglutinin neuraminidase HN proteins via directed evolution, leading to improved therapeutic effects. METHODS: Sialic acid-binding sites (H domains) of the HN protein mutant library were generated using error-prone PCR. Variants of the H domain protein were screened by enzyme-linked immunosorbent assay using HCT 116 cancer cell surface molecules. The mutant S519G H domain protein showed the highest affinity for the surface protein of HCT 116 cells compared to that of different types of cancer cells. This showed that the S519G mutant H domain protein gene replaced the same part of the original HN protein gene, and S519G mutant recombinant NDV (rNDV) was constructed and recovered. S519G rNDV cancer cell killing effects were tested using the MTT assay with various cancer cell types, and the tumor suppression effect of the S519G mutant rNDV was tested in a xenograft mouse model implanted with cancer cells, including HCT 116 cells. RESULTS: S519G rNDV showed increased specificity and enhanced killing ability of HCT 116 cells among various cancer cells and a stronger suppressive effect on tumor growth than the original recombinant NDV. Directed evolution using an artificial amino acid change in the NDV HN (S519G mutant) protein increased its specificity and oncolytic effect in colorectal cancer without changing its virulence. CONCLUSION: These results provide a new methodology for the use of directed evolution technology for more effective oncolytic virus development.