N-acetylneuraminic Acid Research Articles

N-Acetylneuraminic acid triggers endothelial pyroptosis and promotes atherosclerosis progression via GLS2-mediated glutaminolysis pathway.

Vascular endothelial injury initiates atherosclerosis (AS) progression. N-Acetylneuraminic acid (Neu5Ac) metabolic disorder was found to intensify endothelial mitochondrial damage. And GLS2-associated glutaminolysis disorder contributed to mitochondrial dysfunction. However, mechanisms underlying Neu5Ac-associated mitochondrial dysfunction as well as its association with GLS2 remains unclear. In this study, we constructed GLS2-/-ApoE-/- mice by using HBLV-GLS2 shRNA injection. And methods like immunofluorescence, western blotting, transmission electron microscopy were applied to detect profiles of endothelial injury and AS progression both in vivo and in vitro. We demonstrated that Neu5Ac accumulation increased GLS2 expression and promoted glutaminolysis disorder, which further induced endothelial mitochondrial dysfunction via a pyroptosis-dependent pathway in vivo and in vitro. Mechanically, Neu5Ac interacted with SIRT3 and led to FOXO3a deacetylation and phosphorylation, further facilitated c-Myc antagonism and ultimately increased GLS2 levels. Inhibition of GLS2 could improve mitochondrial function and mitigate pyroptosis process. In addition, blocking Neu5Ac production using neuraminidases (NEUs) inhibitor could rescue endothelial damage and alleviate AS development in ApoE-/- mice. These findings proposed that Neu5Ac induced GLS2-mediated glutaminolysis disorder and then promoted mitochondrial dysfunction in a pyroptosis-dependent pathway. Targeting GLS2 or inhibiting Neu5Ac production could prevent AS progression.

Screening and Characterization of Sialic Acid-Binding Variable Lymphocyte Receptors from Hagfish

Sialic acid is a diverse group of monosaccharides often found on the termini of N- and O-linked glycans as well as being components of glycoconjugates. Hypersialylation has been associated with the progression of chronic inflammation-mediated diseases such as cardiovascular disease and cancer. Given its role in infection and disease-related processes, sialic acid is a promising target for therapeutic approaches that utilize carbohydrate-binding molecules. In this study, we screened for sialic acid-recognizing variable lymphocyte receptors (VLRBs) or ccombodies from inshore hagfish (Eptatretus burgeri) using a synthetic Neu5Ac-glycoconjugate as an antigen in immunoassay. Resulting ccombodies, 2D8, 5G11, 4A1, and 5F8 were further characterized in terms of their binding activity and specificity. A competitive ELISA using free haptens showed strong inhibition using either N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The half-maximal inhibitory concentrations (IC50) for Neu5Ac ranged from 7.02 to 17.06 mM, with candidates 4A1 and 5G11 requiring the least and highest amounts, respectively. IC50 values for Neu5Gc ranged from 8.12 to 13.91 mM, for 4A1 and 5G11, respectively. Candidate ccombodies also detected naturally occurring sialic acid from known sialoglycoproteins using a dot blot assay. Neu5Gc-5G11 and Neu5Ac-2D8 yielded the strongest and weakest docking interactions with affinity values of −5.9 kcal/mol and −4.9 kcal/mol, respectively. Hydrogen bonding and hydrophobic interactions were predicted to be the predominant noncovalent forces observed between the ccombodies and sialic acid. This study demonstrates that glycan-binding VLRBs from hagfish hold promise in augmenting the glycobiologists’ toolkit in investigating the roles of glycans in human and animal health and disease.

A dual-capture-system polymer based on imprinted cavities and post-imprinting modification sites with significantly improved affinity and specificity for sialic acid and sialylated glycoprotein

The abnormal expression of N-acetylneuraminic acid (SA) and sialylated glycoproteins in biological fluids are closely associated with various diseases including cancer. However, the low content of SA and the strong interference of complex matrix greatly influence the effective capture of SA in biosamples prior to analysis. Herein, a dual-capture-system strategy based on molecular imprinting and post-imprinting modification (PIM) was proposed to precisely capture SA with improved binding affinity and specificity. After imprinting with SA as template, dynamic imine bonds are introduced by post-imprinting modification, enabling sufficiently high specificity to capture SA through imprinting cavities and the dynamic imine bonds hydrolysis reaction simultaneously. The prepared magnetic PIM polymers (Mag-MIPs-PIM) exhibited significantly high specificity both for SA (IF = 4.24) and sialylated glycoprotein (IFTRF = 3.50). In addition, the feasibility of Mag-MIPs-PIM for practical application was demonstrated by association with HPLC for the determination of SA in human serum, and an LOD of 0.01 × 10−2 g L−1 was obtained. The proposed strategy based on molecular imprinting and PIM provides a new inspiration for the improvement of selectivity of the molecularly imprinted polymers.

Metabolomic alterations in the plasma of patients with various clinical manifestations of COVID-19

BackgroundThe metabolomic profiles of individuals with different clinical manifestations of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection have not been clearly characterized.MethodsWe performed metabolomics analysis of 166 individuals, including 62 healthy controls, 16 individuals with asymptomatic SARS-CoV-2 infection, and 88 patients with moderate (n = 42) and severe (n = 46) symptomatic 2019 coronavirus disease (COVID-19; 17 with short-term and 34 with long-term nucleic-acid test positivity). By examining differential expression, we identified candidate metabolites associated with different SARS-CoV-2 infection presentations. Functional and machine learning analyses were performed to explore the metabolites’ functions and verify their candidacy as biomarkers.ResultsA total of 417 metabolites were detected. We discovered 70 differentially expressed metabolites that may help differentiate asymptomatic infections from healthy controls and COVID-19 patients with different disease severity. Cyclamic acid and N-Acetylneuraminic Acid were identified to distinguish symptomatic infected patients and asymptomatic infected patients. Shikimic Acid, Glycyrrhetinic acid and 3-Hydroxybutyrate can supply significant insights for distinguishing short-term and long-term nucleic-acid test positivity.ConclusionMetabolomic profiling may highlight novel biomarkers for the identification of individuals with asymptomatic SARS-CoV-2 infection and further our understanding of the molecular pathogenesis of COVID-19.

Modelling Approach for the Continuous Biocatalytic Synthesis of N-Acetylneuraminic Acid in Packed Bed Reactors

Continuous flow technologies have become increasingly important for biocatalytic processes. In this study, we present the application and modelling of covalently immobilised N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase in packed bed reactors for the synthesis of N-acetylneuraminic acid. The immobilised enzymes were stable under continuous flow process conditions with half-life times of >28 d (epimerase immobilised on hexamethylamino methacrylate HA403/M) or 58 d (lyase immobilised on dimenthylamino methacrylate ECR8309M), suitable for continuous flow applications. Kinetic studies revealed Michaelis–Menten kinetic behaviour for both enzymes. The kinetic parameters and the inhibitions were analysed under continuous flow conditions and were integrated into a process model using Python. The model was validated by varying flow rates, the mass of immobilised enzymes and the reactor dimensions and shows a low error compared to the measured data. An error accuracy of 6% (epimerase) or 9% (lyase) was achieved. The product concentrations of the enzyme cascade at the end of the packed bed reactor can be predicted with an accuracy of 9% for the calculation of a large column (84.5 mL) or of 24% if several small columns (2.5 mL, 0.8 mL) are connected in series. The developed model has proved to be valid and will be used to optimise the process with respect to substrate concentrations, reactor dimensions and flow rate.

Structural and quantitative characterization of membrane N-glycans from MIN6 mouse pancreatic beta cells using liquid chromatography-quadrupole-Orbitrap tandem mass spectrometry

MIN6, a mouse pancreatic beta cell line, is used in diabetes research, and the cellular N-glycoproteins in membrane are important in regulating the metabolism of insulin secretion. However, the identities of N-glycans in MIN6 cells are yet to be fully elucidated. In this study, the structures of N-glycans were analyzed using liquid chromatography-electrospray ionization-higher energy collisional dissociation-tandem mass spectrometry. The abundances (%) of each N-glycan relative to the total N-glycans (100 %) were also obtained. Fifty N-glycans (with relative abundance of each > 0.5 %) were obtained, revealing 22 bisecting N-acetylglucosamine (GlcNAc; associated with cell adhesion and growth; sum of relative abundance of each: 27.1 %), 21 core-fucosylated (associated with glucose sensing and insulin secretion regulation; 28.3 %), and 16 sialylated (N-acetylneuraminic acid; related to the expression of glucose transporters and diabetes;15.5 %) N-glycans. Membranes contained higher bisecting GlcNAc and core-fucosylation, similar sialylation, but less high-mannosylation than the lysate (the cellular contents). Notably, all bisecting GlcNAc N-glycans were categorized into structures with (16.6 %) or without (10.5 %) core-fucosylation and with (6.9 %) or without (20.2 %) sialylation. The bisecting GlcNAc structures were not found in human islets; moreover, sialylation levels were 6.9 times higher than for human islets. These structural characteristics of N-glycans affect their cell adhesion and distribution through homologous interactions between beta cells, leading to increased insulin secretion efficiency. This study is the first to identify the structures and quantities of 50 N-glycans in MIN6 cell membranes that may play an important role in regulating the functions of pancreatic beta cells.

Elevated muramyl dipeptide by sialic acid-facilitated postantibiotic pathobiont expansion contributes to gut dysbiosis-induced mastitis in mice

IntroductionIn responses to antibiotics exposure, gut dysbiosis is a risk factor not only for pathogen infection but also for facilitating pathobiont expansion, resulting in increased inflammatory responses in the gut and distant organs. However, how this process is regulated has not been fully elucidated. ObjectivesIn this study, we investigated the role of sialic acid, a host-derived carbohydrate, in the pathogenesis of gut dysbiosis-derived inflammation in distant organs. MethodsAmpicillin (Amp)-induced gut dysbiotic mice were treated with N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) for three weeks to assess the role of sialic acids in mastitis. The underlying mechanism by which sialic acids regulate mastitis was explored using 16S rRNA sequencing, transcriptomics and employed multiple molecular approaches. ResultsAdministration of Neu5Ac and Neu5Gc exacerbated gut dysbiosis-induced mastitis and systemic inflammation. The gut dysbiosis caused by Amp was also aggravated by sialic acid. Notably, increased Enterococcus expansion, which was positively correlated with inflammatory markers, was observed in both Neu5Ac- and Neu5Gc-treated gut dysbiotic mice. Treatment of mice with Enterococcus cecorum (E. cecorum) aggravated gut dysbiosis-induced mastitis. Mechanically, sialic acid-facilitated E. cecorum expansion promoted muramyl dipeptide (MDP) release, which induced inflammatory responses by activating the NOD2-RIP2-NF-κB axis. ConclusionsCollectively, our data reveal a role of sialic acid-facilitated postantibiotic pathobiont expansion in gut dysbiosis-associated inflammation, highlighting a potential strategy for disease prevention by regulating the MDP-NOD2-RIP2 axis.

Dual-emission rare-earth fluorescent nanomaterials for ratiometric and visual detection of N-acetylneuraminic acid and applications in information encryption and anti-counterfeiting

N-acetylneuraminic acid (NANA) can be used as a biomarker for many types of cancers. Currently, there are various methods for detecting NANA but showing some shortcomings that limit the real-time diagnosis of cancer. In contrast, fluorescence analysis has obvious advantages such as low cost, fast response time, and easy operation, and it also enables visual detection for real-time cancer monitoring. Therefore, the establishment of an efficient and rapid detection method is essential for the early prevention and treatment of the disease. Based on the properties of layered rare-earth hydroxide (LRH), we synthesized a dual-emission fluorescent material (NDC/SDS-LEuH), and further fabricated a fluorescent nanoprobe (ANP) for the detection of NANA. The probe has the advantages of high sensitivity (LOD = 32.9 μM) and high selectivity with fast response. During the sensing process, the dual emission of the probe shows opposite changes due to the photoinduced electron transfer (PET) effect and the interaction between NANA and the probe. The color changes of the system can be observed under UV irradiation. Therefore, a visual platform was developed to detect NANA with a LOD of 0.09 mM. In addition, a probe hydrogel was prepared, which can be applied in the anti-counterfeiting to improve the difficulty of counterfeiting and the security of anti-counterfeiting. The probe achieves ratiometric fluorescence detection of NANA, which reduces background interference and improves the accuracy of detection. A visual detection platform was fabricated to realize the real-time detection. In addition, the prepared probe hydrogel showed the potential applications in anti-counterfeiting, which provided new ideas for the design and application of anti-counterfeiting materials.

Glycoengineering with neuraminic acid analogs to label lipooligosaccharides and detect native sialyltransferase activity in gram-negative bacteria.

Lipooligosaccharides (LOS) are the most abundant cell surface glycoconjugates on the outer membrane of Gram-negative bacteria. They play important roles in host-microbe interactions. Certain Gram-negative pathogenic bacteria cap their LOS with the sialic acid, N-acetylneuraminic acid (Neu5Ac), to mimic host glycans that among others protects these bacteria from recognition by the hosts immune system. This process of molecular mimicry is not fully understood and remains under investigated. To explore the functional role of sialic acid-capped lipooligosaccharides (LOS) at the molecular level, it is important to have tools readily available for the detection and manipulation of both Neu5Ac on glycoconjugates and the involved sialyltransferases, preferably in live bacteria. We and others have shown that the native sialyltransferases of some Gram-negative bacteria can incorporate extracellular unnatural sialic acid nucleotides onto their LOS. We here report on the expanded use of native bacterial sialyltransferases to incorporate neuraminic acids analogs with a reporter group into the LOS of a variety of Gram-negative bacteria. We show that this approach offers a quick strategy to screen bacteria for the expression of functional sialyltransferases and the ability to use exogenous CMP-Neu5Ac to decorate their glycoconjugates. For selected bacteria we also show this strategy complements two other glycoengineering techniques, Metabolic Oligosaccharide Engineering (MOE) and Selective Exo-Enzymatic Labeling (SEEL), and that together they provide tools to modify, label, detect and visualize sialylation of bacterial LOS.

Integrating Deep Learning and Synthetic Biology: A Co-Design Approach for Enhancing Gene Expression via N-Terminal Coding Sequences.

N-terminal coding sequence (NCS) influences gene expression by impacting the translation initiation rate. The NCS optimization problem is to find an NCS that maximizes gene expression. The problem is important in genetic engineering. However, current methods for NCS optimization such as rational design and statistics-guided approaches are labor-intensive yield only relatively small improvements. This paper introduces a deep learning/synthetic biology codesigned few-shot training workflow for NCS optimization. Our method utilizes k-nearest encoding followed by word2vec to encode the NCS, then performs feature extraction using attention mechanisms, before constructing a time-series network for predicting gene expression intensity, and finally a direct search algorithm identifies the optimal NCS with limited training data. We took green fluorescent protein (GFP) expressed by Bacillus subtilis as a reporting protein of NCSs, and employed the fluorescence enhancement factor as the metric of NCS optimization. Within just six iterative experiments, our model generated an NCS (MLD62) that increased average GFP expression by 5.41-fold, outperforming the state-of-the-art NCS designs. Extending our findings beyond GFP, we showed that our engineered NCS (MLD62) can effectively boost the production of N-acetylneuraminic acid by enhancing the expression of the crucial rate-limiting GNA1 gene, demonstrating its practical utility. We have open-sourced our NCS expression database and experimental procedures for public use.

Neu5Gc binding loss of subtype H7 influenza A virus facilitates adaptation to gallinaceous poultry following transmission from waterbirds.

Between 2013 and 2018, the novel A/Anhui/1/2013 (AH/13)-lineage H7N9 virus caused at least five waves of outbreaks in humans, totaling 1,567 confirmed human cases in China. Surveillance data indicated a disproportionate distribution of poultry infected with this AH/13-lineage virus, and laboratory experiments demonstrated that this virus can efficiently spread among chickens but not among Pekin ducks. The underlying mechanism of this selective transmission remains unclear. In this study, we demonstrated the absence of Neu5Gc expression in chickens across all respiratory and gastrointestinal tissues. However, Neu5Gc expression varied among different duck species and even within the tissues of the same species. The AH/13-lineage viruses exclusively bind to acetylneuraminic acid (Neu5Ac), in contrast to wild waterbird H7 viruses that bind both Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The level of Neu5Gc expression influences H7 virus replication and facilitates adaptive mutations in these viruses. In summary, our findings highlight the critical role of Neu5Gc in affecting the host range and interspecies transmission dynamics of H7 viruses among avian species.IMPORTANCEMigratory waterfowl, gulls, and shorebirds are natural reservoirs for influenza A viruses (IAVs) that can occasionally spill over to domestic poultry, and ultimately humans. This study showed wild-type H7 IAVs from waterbirds initially bind to glycan receptors terminated with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). However, after enzootic transmission in chickens, the viruses exclusively bind to Neu5Ac. The absence of Neu5Gc expression in gallinaceous poultry, particularly chickens, exerts selective pressure, shaping IAV populations, and promoting the acquisition of adaptive amino acid substitutions in the hemagglutinin protein. This results in the loss of Neu5Gc binding and an increase in virus transmissibility in gallinaceous poultry, particularly chickens. Consequently, the transmission capability of these poultry-adapted H7 IAVs in wild water birds decreases. Timely intervention, such as stamping out, may help reduce virus adaptation to domestic chicken populations and lower the risk of enzootic outbreaks, including those caused by IAVs exhibiting high pathogenicity.

Structural and quantitative comparison of viral infection-associated N-glycans in plasma from humans, pigs, and chickens: Greater similarity between humans and chickens than pigs

Host N-glycans play an essential role in the attachment, invasion, and infection processes of viruses, including zoonotic infectious diseases. The similarity of N-glycans in the trachea and lungs of humans and pigs facilitates the cross-species transmission of influenza viruses through respiratory tracts. In this study, the structure and quantity of N-glycans in the plasma of humans, pigs, and chickens were analyzed using liquid chromatography-quadrupole-Orbitrap-tandem mass spectrometry. N-glycans in humans (35), pigs (28), and chickens (53) were identified, including the most abundant, species-common, and species-specific N-glycans. Among the N-glycans (relative quantity >0.5%), the sialic acid derivative of N-acetylneuraminic acid was identified in humans (the sum of the relative quantities of each; 64.3%), pigs (45.5%), and chickens (64.4%), whereas N-glycolylneuraminic acid was only identified in pigs (18.1%). Sialylated N-glycan linkage isomers are the influenza virus receptors (α2-6 in humans, α2-3 and α2-6 in pigs, and α2-3 in chickens). Only α2-6 linkages (human, 58.2%; pig, 44.8%; and chicken, 60.6%) were more abundant than α2-3/α2-6 linkages (human, 4.6%; pig, 0.6%; and chicken, 3.4%) and only α2-3 linkages (human, 1.5%; pig, 0.1%; and chicken, 0.4%). Fucosylation, which can promote viral infection through immune modulation, was more abundant in pigs (76.1%) than in humans (36.4%) and chickens (16.7%). Bisecting N-acetylglucosamine, which can suppress viral infection by inhibiting sialylation, was identified in humans (10.3%) and chickens (16.9%), but not in pigs. These results indicate that plasma N-glycans are similar in humans and chickens. This is the first study to compare plasma N-glycans in humans, pigs, and chickens.

Enzymatic promiscuity and underground reactions accounted for the capability of Escherichia coli to use the non-natural chemical synthon 2,4-dihydroxybutyric acid as a carbon source for growth

2,4-dihydroxybutyric acid (DHB) and 2-keto-4-hydroxybutyrate (OHB) are non-natural molecules obtained through synthetic pathways from renewable carbon source. As they are structurally similar to lactate and pyruvate respectively, they could possibly interfere with the metabolic network of Escherichia coli. In fact, we showed that DHB can be easily oxidized by the membrane associated L and D-lactate dehydrogenases encoded by lldD, dld and ykgF into OHB, and the latter being cleaved into pyruvate and formaldehyde by several pyruvate-dependent aldolases, with YagE being the most effective. While formaldehyde was readily detoxified into formate, Escherichia coli K12 MG1655 strain failed to grow on DHB despite of the production of pyruvate. To find out the reason for this failure, we constructed a mutant strain whose growth was rendered dependent on DHB and subjected this strain to adaptive evolution. Genome sequencing of the adapted strain revealed an essential role for ygbI encoding a transcriptional repressor of the threonate operon in this DHB-dependent growth. This critical function was attributed to the derepression of ygbN encoding a putative threonate transporter, which was found to exclusively transport the D form of DHB. A subsequent laboratory evolution was carried out with E. coli K12 MG1655 deleted for ΔygbI to adapt for growth on DHB as sole carbon source. Remarkably, only two additional mutations were disclosed in the adapted strain, which were demonstrated by reverse engineering to be necessary and sufficient for robust growth on DHB. One mutation was in nanR encoding the transcription repressor of sialic acid metabolic genes, causing 140-fold increase in expression of nanA encoding N-acetyl neuraminic acid lyase, a pyruvate-dependent aldolase, and the other was in the promoter of dld leading to 14-fold increase in D-lactate dehydrogenase activity on DHB. Taken together, this work illustrates the importance of promiscuous enzymes in underground metabolism and moreover, in the frame of synthetic pathways aiming at producing non-natural products, these underground reactions could potentially penalize yield and title of these bio-based products.

Aeromonas salmonicida AI-1 and AI-2 quorum sensing pathways are differentially regulated by rainbow trout mucins and during in vivo colonization

Aeromonas salmonicida is an opportunistic pathogen with relevance for aquaculture. Fish epithelia are covered by a mucus layer, composed mainly by highly glycosylated mucins, which are the first point of contact between fish and pathogens. Quorum sensing (QS), a bacterial communication mechanism through secreted autoinducer signals that governs gene expression, influences bacterial growth and virulence. The main A. salmonicida autoinducers are mediated by the luxS and asaI genes, corresponding to inter- and intraspecies communication, respectively. The aim of this study was to determine the effect of the mucins that pathogens encounter during colonization of the gill and skin on A. salmonicida QS. We found that expression of A. salmonicida asaI, but not luxS, was increased after culture at 20 °C compared to 10 °C. Rainbow trout gill and skin mucins up-regulated asaI expression 2-fold but down-regulated luxS 10-fold. The downregulation of luxS was reflected by a reduction in autoinducer-2 secretion. Mucins isolated from skin had a stronger inhibitory effect than mucins isolated from gills on both luxS expression and A1-2 secretion, consistent with a higher relative abundance of N-Acetylneuraminic acid on skin mucins than on gill mucins. Reduction of AI-2 production by mucins or luxS-deletion lead to a reduced A. salmonicida auto-aggregation. Furthermore, after colonization of the gill, luxS was down regulated whereas asaI expression was upregulated. Both in vivo and in vitro, the expression of luxS and asaI were thus differentially regulated, frequently in an inverse manner. The strong AI-2 inhibiting effect of the skin mucins is likely part of the mucin-based defense against pathogens.

Dysregulation of cerebrospinal fluid metabolism profiles in spinal muscular atrophy patients: a case control study

BackgroundSpinal muscular atrophy (SMA) is a neurodegenerative disorder. Although prior studies have investigated the metabolomes of SMA in various contexts, there is a gap in research on cerebrospinal fluid (CSF) metabolomics compared to healthy controls. CSF metabolomics can provide insights into central nervous system function and patient outcomes. This study aims to investigate CSF metabolite profiles in untreated SMA patients to enhance our understanding of SMA metabolic dysregulation.MethodsThis case control study included 15 SMA patients and 14 control subjects. CSF samples were collected, and untargeted metabolomics was conducted to detect metabolites in SMA and control groups.ResultsA total of 118 metabolites abundance were significantly changed between the SMA and control groups. Of those, 27 metabolites with variable importance for the projection (VIP) ≥ 1.5 were identified. The top 5 differential metabolites were N-acetylneuraminic acid (VIP = 2.38, Fold change = 0.43, P = 5.49 × 10–5), 2,3-dihydroxyindole (VIP = 2.33, Fold change = 0.39, P = 1.81 × 10–4), lumichrome (VIP = 2.30, Fold change = 0.48, P = 7.90 × 10–5), arachidic acid (VIP = 2.23, Fold change = 10.79, P = 6.50 × 10–6), and 10-hydroxydecanoic acid (VIP = 2.23, Fold change = 0.60, P = 1.44 × 10–4). Cluster analysis demonstrated that the differentially metabolites predominantly clustered within two main categories: protein and amino acid metabolism, and lipid metabolism.ConclusionsThe findings highlight the complexity of SMA, with widespread effects on multiple metabolic pathways, particularly in amino acid and lipid metabolism. N-acetylneuraminic acid may be a potential treatment for functional improvement in SMA. The exact mechanisms and potential therapeutic targets associated with metabolic dysregulation in SMA require further investigation.

Harnessing the acceptor substrate promiscuity of Clostridium botulinum Maf glycosyltransferase to glyco-engineer mini-flagellin protein chimeras

Several bacterial flagellins are O-glycosylated with nonulosonic acids on surface-exposed Serine/Threonine residues by Maf glycosyltransferases. The Clostridium botulinum Maf glycosyltransferase (CbMaf) displays considerable donor substrate promiscuity, enabling flagellin O-glycosylation with N-acetyl neuraminic acid (Neu5Ac) and 3-deoxy-D-manno-octulosonic acid in the absence of the native nonulosonic acid, a legionaminic acid derivative. Here, we have explored the sequence/structure attributes of the acceptor substrate, flagellin, required by CbMaf glycosyltransferase for glycosylation with Neu5Ac and KDO, by co-expressing C. botulinum flagellin constructs with CbMaf glycosyltransferase in an E. coli strain producing cytidine-5’-monophosphate (CMP)-activated Neu5Ac, and employing intact mass spectrometry analysis and sialic acid-specific flagellin biotinylation as readouts. We found that CbMaf was able to glycosylate mini-flagellin constructs containing shortened alpha-helical secondary structural scaffolds and reduced surface-accessible loop regions, but not non-cognate flagellin. Our experiments indicated that CbMaf glycosyltransferase recognizes individual Ser/Thr residues in their local surface-accessible conformations, in turn, supported in place by the secondary structural scaffold. Further, CbMaf glycosyltransferase also robustly glycosylated chimeric proteins constructed by grafting cognate mini-flagellin sequences onto an unrelated beta-sandwich protein. Our recombinant engineering experiments highlight the potential of CbMaf glycosyltransferase in future glycoengineering applications, especially for the neo-O-sialylation of proteins, employing E. coli strains expressing CMP-Neu5Ac (and not CMP-KDO).

Development and evaluation of an immunoassay for the quantification of N-acetylneuraminic acid (Neu5Ac) in foods and biosamples

N-acetylneuraminic acid is an active ingredient in tonic foods and an important additive in foods and biopharmaceuticals. To address the limitations of existing methods of N-acetylneuraminic acid quantification, we developed an immunoassay based on antibodies induced in hens using artificial antigen, showing high sensitivity and specificity with no cross-reactivity with eight N-acetylneuraminic acid analogues. An IgY-based indirect competitive enzyme-linked immunosorbent assay showed a detection range of 1.14 to 70.08 ng/mL and a limit of detection of 0.57 ng/mL. In spiked samples, recoveries by the indirect competitive enzyme-linked immunosorbent assay ranged from 74.05% to 110.87% compared with HPLC (73.01% to 108.8%). Consistency between the indirect competitive enzyme-linked immunosorbent assay and HPLC was satisfactory (R2 = 0.9736), demonstrating this established immunoassay as a rapid and reliable approach for N-acetylneuraminic acid analysis. The assay described in this study provides an important method for the screening of N-acetylneuraminic acid in biological samples and foodstuffs.

Chemical-induced hypoxia injury of syncitialized BeWo cells promotes a reduction of terminal N-acetylneuraminic acid and mannose

Chemical-induced hypoxia injury of syncitialized BeWo cells promotes a reduction of terminal N-acetylneuraminic acid and mannose

Novel sialidase inhibitors suppress mumps virus replication and infection.

The prevalent human pathogen, mumps virus (MuV; orthorubulavirus parotitidis) causes various complications and serious sequelae, such as meningitis, encephalitis, deafness, and impaired fertility. Direct-acting antivirals (DAAs) targeting MuV which can prevent mumps and mumps-associated complications and sequelae are yet to be developed. Paramyxoviridae family members, such as MuV, possess viral surface hemagglutinin-neuraminidase (HN) protein with sialidase activity which facilitates efficient viral replication. Therefore, to develop DAAs targeting MuV we synthesized MuV sialidase inhibitors. It is proposed that the viral HN has a single functional site for N-acetylneuraminic acid (Neu5Ac) binding and sialidase activity. Further, the known MuV sialidase inhibitor is an analog of Neu5Ac-2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA)-which lacks potency. DANA derivatives with higher MuV sialidase inhibitory potency are lacking. The MuV-HN-Neu5Ac binding site has a hydrophobic cavity adjacent to the C4 position of Neu5Ac. Exploiting this, here, we synthesized DANA derivatives with increasing hydrophobicity at its C4 position and created 3 novel sialidase inhibitors (Compounds 1, 2, and 3) with higher specificity for MuV-HN than DANA; they inhibited MuV replication step to greater extent than DANA. Furthermore, they also inhibited hemagglutination and the MuV infection step. The insight-that these 3 novel DANA derivatives possess linear hydrocarbon groups at the C4-hydroxyl group of DANA-could help develop highly potent sialidase inhibitors with high specificity for MuV sialidase, which may function as direct-acting MuV-specific antivirals.

Impact of Food on the Oral Absorption of N-Acetyl-D-Mannosamine in Healthy Men and Women.

N-Acetyl-D-mannosamine (ManNAc) is an endogenous monosaccharide and precursor of N-acetylneuraminic acid (Neu5Ac), a critical sialic acid. ManNAc is currently under clinical development to treat GNE myopathy, a rare muscle-wasting disease. In this randomized, open-label, 2-sequence, crossover study, 16 healthy women and men were administered a single oral dose of ManNAc under fasting and fed conditions. Blood samples were collected for 48hours after dosing for quantification of plasma ManNAc and Neu5Ac concentrations. Noncompartmental pharmacokinetic and deconvolution analyses were performed using baseline-corrected plasma concentration data. Administration of ManNAc in the fed state resulted in a 1.6-fold increase in ManNAc exposure, compared to fasting conditions. A concurrent increase in Neu5Ac exposure was observed in the presence of food. Deconvolution analysis indicated that the findings were attributed to prolonged absorption rather than an enhanced rate of absorption. The impact of food on ManNAc pharmacokinetics was greater in women than men (fed/fasted area under the concentration-time curve from time 0 to infinity mean ratio: 198% compared to 121%). It is hypothesized that the presence of food slows gastric emptying, allowing a gradual release of ManNAc into the small intestine, translating into improved ManNAc absorption. The results suggest that taking ManNAc with food may enhance its therapeutic activity and/or reduce the daily dosage requirement.