Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.
N-Acetylneuraminic Acid/metabolism* ; Bacillus subtilis/metabolism* ; Promoter Regions, Genetic/genetics* ; Binding Sites ; Biosensing Techniques

A boy, aged 3 hours, was admitted due to a prenatal diagnosis of fetal hydrops at 3 hours after resuscitation for birth asphyxia. Prenatal examination at 5 months of gestation showed massive ascites in the fetus, and after birth, the boy had the manifestations of systemic hydroderma, massive ascites, coarse face, and hepatomegaly. Genetic testing revealed heterozygous mutations in the SLC17A5 gene, and there was a significant increase in urinary free sialic acid. Placental pathology showed extensive vacuolization in villous stromal cells, Hofbauer cells, cytotrophoblast cells, and syncytiotrophoblast cells in human placental chorionic villi. The boy was finally diagnosed with free sialic acid storage disorders (FSASDs). This is the first case of FSASDs with the initial symptom of fetal hydrops reported in China. The possibility of FSASDs should be considered for cases with non-immune hydrops fetalis, and examinations such as placental pathology and urinary free sialic acid may help with early diagnosis and clinical decision making.
Infant, Newborn ; Male ; Humans ; Female ; Pregnancy ; Hydrops Fetalis/genetics* ; N-Acetylneuraminic Acid ; Placenta/pathology* ; Ascites

Immunoglobulin (IgG) glycosylation affects the effector functions of IgG in a myriad of biological processes and has been closely associated with numerous autoimmune diseases, including systemic lupus erythematosus (SLE), thus underlining the pathogenic role of glycosylation aberration in autoimmunity. This study aims to explore the relationship between IgG sialylation patterns and lupus pregnancy. Relative to that in serum samples from the control cohort, IgG sialylation level was aberrantly downregulated in serum samples from the SLE cohort at four stages (from preconception to the third trimester of pregnancy) and was significantly associated with lupus activity and fetal loss during lupus pregnancy. The type I interferon signature of pregnant patients with SLE was negatively correlated with the level of IgG sialylation. The lack of sialylation dampened the ability of IgG to suppress the functions of plasmacytoid dendritic cells (pDCs). RNA-seq analysis further revealed that the expression of genes associated with the spleen tyrosine kinase (SYK) signaling pathway significantly differed between IgG- and deSia-IgG-treated pDCs. This finding was confirmed by the attenuation of the ability to phosphorylate SYK and BLNK in deSia-IgG. Finally, the coculture of pDCs isolated from pregnant patients with SLE with IgG/deSia-IgG demonstrated the sialylation-dependent anti-inflammatory function of IgG. Our findings suggested that IgG influences lupus activity through regulating pDCs function via the modulation of the SYK pathway in a sialic acid-dependent manner.
Humans ; Pregnancy ; Female ; Lupus Erythematosus, Systemic/pathology* ; Signal Transduction ; N-Acetylneuraminic Acid/metabolism* ; Immunoglobulin G ; Dendritic Cells/pathology*

Lectins are proteins responsible for recognizing the signals of sugar molecules in the body. Sialic acid-binding immunoglobulin-like lectins (Siglecs) regulate the innate and adaptive immune responses in the tumor microenvironment by recognizing the glycan structure containing sialic acid and mediating downstream signals through immune receptor tyrosine inhibitory motifs. In recent years, a variety of tumor treatment strategies targeting the sialic acid-Siglecs axis have been introduced, including sialoglycoprotein-mediated drug delivery and antibody mediated inhibition of Siglecs from recognizing tumor surface ligands. In the future, by combining with glycoprotein nanotherapy, antibody therapy and gene therapy, Siglecs can be used to accurately locate tumor targets and release the anti-tumor immunity, so as to achieve the purpose of effective cure of tumors.
Sialic Acid Binding Immunoglobulin-like Lectins/metabolism* ; N-Acetylneuraminic Acid ; Immunoglobulins/metabolism* ; Receptors, Immunologic ; Ligands

Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker's yeast were added to the above fermentation broth to synthesize 3'-sialyllactose (3'-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker's yeast, 20 mmol/L Mg²⁺, the maximum yield of 3'-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3'-SL.
Child ; Humans ; Child, Preschool ; N-Acetylneuraminic Acid ; Escherichia coli/genetics* ; Lactose ; Fermentation ; Saccharomyces cerevisiae ; Oligosaccharides ; Glucosamine

Chronic obstructive pulmonary disease (COPD) has become the third-leading cause of death worldwide, which is a severe economic burden to the healthcare system. Chronic bronchitis is the most common condition that contributes to COPD, both locally and systemically. Neutrophilic inflammation predominates in the COPD airway wall and lumen. Logically, repression of neutrophilia is an essential fashion to COPD treatment. However, currently available anti-neutrophilic therapies provide little benefit in COPD patients and may have serious side effects. Thus, there is an urgent need to explore an effective and safe anti-neutrophilic approach that might delay progression of the disease. Sialic acid-binding immunoglobulin-like lectin (Siglec)-9 is a member of the Siglec cell surface immunoglobulin family. It is noteworthy that Siglec-9 is highly expressed on human neutrophils and monocytes. Ligation of Siglec-9 by chemical compounds or synthetic ligands induced apoptosis and autophagic-like cell death in human neutrophils. Furthermore, administration of antibody to Siglec-E, mouse functional ortholog of Siglec-9, restrained recruitment and activation of neutrophils in mouse models of airway inflammation in vivo. Given the critical role that neutrophils play in chronic bronchitis and emphysema, targeting Siglec-9 could be beneficial for the treatment of COPD, asthma, fibrosis, and related chronic inflammatory lung diseases.
Animals ; Asthma ; Humans ; Lung ; Mice ; N-Acetylneuraminic Acid ; Neutrophils ; Pulmonary Disease, Chronic Obstructive/drug therapy* ; Sialic Acid Binding Immunoglobulin-like Lectins

Neuraminidase (NA) cleaves the glycosidic bond linkages of sialic acids to release the mature virions from infected cells and has been an attractive therapeutic target for anti-influenza agents. In our ongoing investigation of NA inhibitors in mushroom extracts, we found that the extract the fruiting body of Glaziella splendens potently inhibited neuraminidase. The fruiting bodies of G. splendens were extracted and partitioned successively with hexane, ethyl acetate, and butanol. The ethyl acetate soluble-layer was subjected to silica gel and Sephadex LH-20 column chromatographies, and MPLC to obtain five compounds (1–5). Their structures were determined by spectroscopic methods. NA inhibitory activity of these compounds was evaluated using NAs from recombinant rvH1N1, H3N2, and H5N1 influenza A viruses. One compound (1) was elucidated as a new azaphilone derivative, and four compounds (2–5) were identified as entonaemin A, comazaphilone D, rubiginosin A, and entonaemin B, respectively. Compounds 3 and 4 showed considerable inhibitory activity against three types of neuraminidases with the IC₅₀ values of 30.9, 41.8, and 35.7 µM for 3 and 46.5, 50.4, and 29.9 µM for 4, respectively. This study reveals that the fruiting bodies of G. splendens possess azaphilone derivatives with the NA inhibitory activity. This is the first report on the isolation of neuraminidase inhibitors from the fruiting bodies of G. splendens.
Agaricales ; Chromatography ; Fruit ; Influenza A virus ; N-Acetylneuraminic Acid ; Neuraminidase ; Sialic Acids ; Silica Gel ; Virion

OBJECTIVE: Sialic acid (Sia) is an essential nutrient for brain development, learning, memory and cognition and plays a role in neurodevelopment of infants. The aim of this study was to determine whether Sia levels are significantly associated with the autism spectrum disorder (ASD). METHODS: Forty-six ASD children and 30 typically developing children aged 3 to 10 years were included in the study. Behavioral symptoms in ASD children was assessed by the Autism Behavior Checklist (AuBC), the Childhood Autism Rating Scale, and the Aberrant Behavior Checklist (ABC). After the collection of saliva samples, the supernatant was separated. All the samples kept at −80°C until Sia analysis was done. RESULTS: Sia level was found to be significantly lower in the ASD group when compared to healthy controls (p = 0.013). There was no correlation between severity of ASD and salivary Sia levels. We found a negative correlation between AuBC scores and Sia levels and a negative correlation in both ABC Stereotypic Behavior and Hyperactivity/Noncompliance subscales with Sia levels in ASD group. CONCLUSION: The obtained data indicate that Sia levels could have an effect on autism-like behaviors, particularly on stereotypes and hyperactivity.
Autism Spectrum Disorder ; Autistic Disorder ; Behavioral Symptoms ; Brain ; Checklist ; Child ; Cognition ; Humans ; Infant ; Learning ; Memory ; N-Acetylneuraminic Acid ; Saliva

BACKGROUND/OBJECTIVES: Helicobacter pylori (H. pylori) colonization of the stomach mucosa and duodenum is the major cause of acute and chronic gastroduodenal pathology in humans. Efforts to find effective anti-bacterial strategies against H. pylori for the non-antibiotic control of H. pylori infection are urgently required. In this study, we used whey to prepare glycomacropeptide (GMP), from which sialic acid (G-SA) was enzymatically isolated. We investigated the anti-bacterial effects of G-SA against H. pylori in vitro and in an H. pylori-infected murine model. MATERIALS/METHODS: The anti-bacterial activity of G-SA was measured in vitro using the macrodilution method, and interleukin-8 (IL-8) production was measured in H. pylori and AGS cell co-cultures by ELISA. For in vivo study, G-SA 5 g/kg body weight (bw)/day and H. pylori were administered to mice three times over one week. After one week, G-SA 5 g/kg bw/day alone was administered every day for one week. Tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and IL-10 levels were measured by ELISA to determine the anti-inflammatory effects of G-SA. In addition, real-time PCR was performed to measure the genetic expression of cytotoxin-associated gene A (cagA). RESULTS: G-SA inhibited the growth of H. pylori and suppressed IL-8 production in H. pylori and in AGS cell co-cultures in vitro. In the in vivo assay, administration of G-SA reduced levels of IL-1β and IL-6 pro-inflammatory cytokines whereas IL-10 level increased. Also, G-SA suppressed the expression of cagA in the stomach of H. pylori-infected mice. CONCLUSION: G-SA possesses anti-H. pylori activity as well as an anti-H. pylori-induced gastric inflammatory effect in an experimental H. pylori-infected murine model. G-SA has potential as an alternative to antibiotics for the prevention of H. pylori infection and H. pylori-induced gastric disease prevention.
Animals ; Anti-Bacterial Agents ; Body Weight ; Coculture Techniques ; Colon ; Cytokines ; Duodenum ; Enzyme-Linked Immunosorbent Assay ; Helicobacter pylori ; Helicobacter* ; Humans ; In Vitro Techniques ; Interleukin-10 ; Interleukin-6 ; Interleukin-8 ; Methods ; Mice ; Mucous Membrane ; N-Acetylneuraminic Acid* ; Necrosis ; Pathology ; Real-Time Polymerase Chain Reaction ; Stomach ; Stomach Diseases ; Whey

The influenza A (H1N1) virus, also known as swine flu is a leading cause of morbidity and mortality since 2009. There is a need to explore novel anti-viral drugs for overcoming the epidemics. Traditionally, different plant extracts of garlic, ginger, kalmegh, ajwain, green tea, turmeric, menthe, tulsi, etc. have been used as hopeful source of prevention and treatment of human influenza. The H1N1 virus contains an important glycoprotein, known as neuraminidase (NA) that is mainly responsible for initiation of viral infection and is essential for the life cycle of H1N1. It is responsible for sialic acid cleavage from glycans of the infected cell. We employed amino acid sequence of H1N1 NA to predict the tertiary structure using Phyre2 server and validated using ProCheck, ProSA, ProQ, and ERRAT server. Further, the modelled structure was docked with thirteen natural compounds of plant origin using AutoDock4.2. Most of the natural compounds showed effective inhibitory activity against H1N1 NA in binding condition. This study also highlights interaction of these natural inhibitors with amino residues of NA protein. Furthermore, among 13 natural compounds, theaflavin, found in green tea, was observed to inhibit H1N1 NA proteins strongly supported by lowest docking energy. Hence, it may be of interest to consider theaflavin for further in vitro and in vivo evaluation.
Amino Acid Sequence ; Curcuma ; Garlic ; Ginger ; Glycoproteins ; Hope ; In Vitro Techniques ; Influenza A virus ; Influenza A Virus, H1N1 Subtype ; Influenza, Human* ; Life Cycle Stages ; Molecular Docking Simulation ; Mortality ; N-Acetylneuraminic Acid ; Neuraminidase* ; Phytochemicals ; Plant Extracts ; Plants ; Polysaccharides ; Swine ; Tea