Influenza virus and pneumococcal neuraminidases enhance catalysis by similar yet distinct sialic acid-binding strategies.

Influenza A viruses and the bacterium Streptococcus pneumoniae (pneumococci) both express neuraminidases that catalyze release of sialic acid residues from oligosaccharides and glycoproteins. Although these respiratory pathogen neuraminidases function in a similar environment, it remains unclear if these enzymes use similar mechanisms for sialic acid cleavage. Here, we compared the enzymatic properties of neuraminidases from two influenza A subtypes (N1 and N2) and the pneumococcal strain TIGR4 (NanA, NanB, and NanC). Insect cell-produced N1 and N2 tetramers exhibited calcium-dependent activities and stabilities that varied with pH. In contrast, E. coli-produced NanA, NanB, and NanC were isolated as calcium insensitive monomers with stabilities that were more resistant to pH changes. Using a synthetic substrate (MUNANA), all neuraminidases showed similar pH optimums (pH 6-7) that were primarily defined by changes in catalytic rate rather than substrate binding affinity. Upon using a multivalent substrate (fetuin sialoglycans), much higher specific activities were observed for pneumococcal neuraminidases that contain an additional lectin domain. In virions, N1 and especially N2 also showed enhanced specific activity toward fetuin that was lost upon the addition of detergent, indicating the sialic acid-binding capacity of neighboring hemagglutinin molecules likely contributes to catalysis of natural multivalent substrates. These results demonstrate that influenza and pneumococcal neuraminidases have evolved similar yet distinct strategies to optimize their catalytic activity.

Analysis of the intramolecular 1,7-lactone of N-acetylneuraminic acid using HPLC-MS: relationship between detection and stability.

A subclass of the sialic acid family consists of intramolecular lactones that may function as key indicators of physiological and pathological states. However, the existence of these compounds in free form is highly improbable, since they are unlikely to exist in an aqueous solution due to their lability. Current analytical method used to detect them in biological fluids has not recognized their reactivity in solution and is prone to misidentification. However, recent advances in synthetic methods for 1,7-lactones have allowed the preparation of these sialic acid derivatives as authentic reference standards. We report here the development of a new HPLC-MS method for the simultaneous detection of the 1,7-lactone of N-acetylneuraminic acid, its γ-lactone derivative, and N-acetylneuraminic acid that overcomes the limitations of the previous analytical procedure for their identification.

The generation of 5-N-glycolylneuraminic acid as a consequence of high levels of reactive oxygen species.

The presence of N-glycolylneuraminic acid (Neu5Gc), a non-human sialic acid in cancer patients, is currently attributed to the consumption of red meat. Excess dietary red meat has been considered a risk factor causing chronic inflammation and for the development of cancers. However, it remains unknown whether Neu5Gc can be generated via a chemical reaction rather than via a metabolic pathway in the presence of high levels of reactive oxygen species (ROS) found in the inflammatory and tumor environments. In this study, the conversion of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc has been assessed in vitro under conditions mimicking the hydroxyl radical-rich humoral environment found in inflammatory and cancerous tissues. As a result, Neu5Gc has been detected via liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, this conversion has also been found to take place in serum biomatrix containing ROS and in cancer cell cultures with induced ROS production.

Changes in the levels of free sialic acid during ex vivo lung perfusion do not correlate with pulmonary function. Experimental model.

BACKGROUND: Ex vivo lung perfusion (EVLP) constitutes a tool with great research potential due to its advantages over in vivo and in vitro models. Despite its important contribution to lung reconditioning, this technique has the disadvantage of incurring high costs and can induce pulmonary endothelial injury through perfusion and ventilation. The pulmonary endothelium is made up of endothelial glycocalyx (EG), a coating of proteoglycans (PG) on the luminal surface. PGs are glycoproteins linked to terminal sialic acids (Sia) that can affect homeostasis with responses leading to edema formation. This study evaluated the effect of two ex vivo perfusion solutions on lung function and endothelial injury. METHODS: We divided ten landrace swine into two groups and subjected them to EVLP for 120 min: Group I (n = 5) was perfused with Steen® solution, and Group II (n = 5) was perfused with low-potassium dextran-albumin solution. Ventilatory mechanics, histology, gravimetry, and sialic acid concentrations were evaluated. RESULTS: Both groups showed changes in pulmonary vascular resistance and ventilatory mechanics (p < 0.05, Student's t-test). In addition, the lung injury severity score was better in Group I than in Group II (p < 0.05, Mann-Whitney U); and both groups exhibited a significant increase in Sia concentrations in the perfusate (p < 0.05 t-Student) and Sia immunohistochemical expression. CONCLUSIONS: Sia, as a product of EG disruption during EVLP, was found in all samples obtained in the system; however, the changes in its concentration showed no apparent correlation with lung function.

The effect of long-term moderate exercise on myocardial metabolome in rats.

Regular moderate physical exercise is beneficial for the cardiovascular system. Our prior study has demonstrated a long-term moderate exercise (4-week of 60-min 74.0% V̇O2max treadmill running) is optimal in protecting from exhaustive exercise-induced cardiac ischemic injury. This study is aimed to investigate the effect of long-term moderate exercise on myocardial metabolome in rats. Thirteen male Sprague-Dawley rats were randomly assigned into the control group (C) and the long-term moderate exercise group (E). The targeted metabolomics of the myocardium was analyzed by ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system. Results showed that the metabolites categories of bile acids (BAs), fatty acids (FAs), and phenylpropanoic acids were significantly decreased. The biosynthesis of unsaturated FAs pathway was significantly downregulated. The altered metabolites in the E Group included decreased FAs (pentadecanoic acid, 10Z-heptadecenoic acid, dihomo-gamma-linolenic acid, docosahexaenoic acid, docosapentaenoic acid, and 10Z-nonadecenoic acid), decreased BAs (chenodeoxycholic acid and beta-muricholic acid), decreased organic acids (glycolic acid and 2-hydroxyglutaric acid), decreased carbohydrate (N-acetylneuraminic acid, Neu5Ac), decreased amino acids (α-aminobutyric acid and norvaline), decreased phenylpropanoic acids (hydroxyphenyllactic acid), and benzoic acids (4-hydroxybenzoic acid and phthalic acid). The results indicated that long-term moderate exercise has promoted lipids utilization in myocardium while exerted little influence on carbohydrate metabolism and diminished many detrimental metabolites. Notably, decrease of myocardial carbohydrate Neu5Ac after long-term moderate exercise might predict a prospective metabolomics biomarker for cardioprotection. This research has displayed the effect of long-term moderate exercise on myocardial metabolomic profiling in rats and indicated some promising metabolites which can be applied for exercise benefits in future.

A novel C-domain-dependent inhibition of the rainbow trout CMP-sialic acid synthetase activity by CMP-deaminoneuraminic acid.

The CMP-sialic acid synthetase (CSS) activates free sialic acid (Sia) to CMP-Sia using CTP, and is prerequisite for the sialylation of cell surface glycoconjugates. The vertebrate CSS consists of two domains, a catalytic N-domain and a non-catalytic C-domain. Although the C-domain is not required for the CSS enzyme to synthesize CMP-Sia, its involvement in the catalytic activity remains unknown. First, the real-time monitoring of CSS-catalyzed reaction was performed by 31P NMR using the rainbow trout CSS (rtCSS). While a rtCSS lacking the C-domain (rtCSS-N) similarly activated both deaminoneuraminic acid (Kdn) and N-acetylneuraminic acid (Neu5Ac), the full-length rtCSS (rtCSS-FL) did not activate Kdn as efficiently as Neu5Ac. These results suggest that the C-domain of rtCSS affects the enzymatic activity, when Kdn was used as a substrate. Second, the enzymatic activity of rtCSS-FL and rtCSS-N was measured under various concentrations of CMP-Kdn. Inhibition by CMP-Kdn was observed only for rtCSS-FL, but not for rtCSS-N, suggesting that the inhibition was C-domain-dependent. Third, the inhibitory effect of CMP-Kdn was also investigated using the mouse CSS (mCSS). However, no inhibition was observed with mCSS even at high concentrations of CMP-Kdn. Taken together, the data demonstrated that the C-domain is involved in the CMP-Kdn-dependent inhibition of rtCSS, which is a novel regulation of the Sia metabolism in rainbow trout.

How bacteria utilize sialic acid during interactions with the host: snip, snatch, dispatch, match and attach.

N -glycolylneuraminic acid (Neu5Gc), and its precursor N-acetylneuraminic acid (Neu5Ac), commonly referred to as sialic acids, are two of the most common glycans found in mammals. Humans carry a mutation in the enzyme that converts Neu5Ac into Neu5Gc, and as such, expression of Neu5Ac can be thought of as a 'human specific' trait. Bacteria can utilize sialic acids as a carbon and energy source and have evolved multiple ways to take up sialic acids. In order to generate free sialic acid, many bacteria produce sialidases that cleave sialic acid residues from complex glycan structures. In addition, sialidases allow escape from innate immune mechanisms, and can synergize with other virulence factors such as toxins. Human-adapted pathogens have evolved a preference for Neu5Ac, with many bacterial adhesins, and major classes of toxin, specifically recognizing Neu5Ac containing glycans as receptors. The preference of human-adapted pathogens for Neu5Ac also occurs during biosynthesis of surface structures such as lipo-oligosaccharide (LOS), lipo-polysaccharide (LPS) and polysaccharide capsules, subverting the human host immune system by mimicking the host. This review aims to provide an update on the advances made in understanding the role of sialic acid in bacteria-host interactions made in the last 5-10 years, and put these findings into context by highlighting key historical discoveries. We provide a particular focus on 'molecular mimicry' and incorporation of sialic acid onto the bacterial outer-surface, and the role of sialic acid as a receptor for bacterial adhesins and toxins.

Sialic acid and food allergies: The link between nutrition and immunology.

Food allergies (FA), a major public health problem recognized by the World Health Organization, affect an estimated 3%-10% of adults and 8% of children worldwide. However, effective treatments for FA are still lacking. Recent advances in glycoimmunology have demonstrated the great potential of sialic acids (SAs) in the treatment of FA. SAs are a group of nine-carbon α-ketoacids usually linked to glycoproteins and glycolipids as terminal glycans. They play an essential role in modulating immune responses and may be an effective target for FA intervention. As exogenous food components, sialylated polysaccharides have anti-FA effects. In contrast, as endogenous components, SAs on immunoglobulin E and immune cell surfaces contribute to the pathogenesis of FA. Given the lack of comprehensive information on the effects of SAs on FA, we reviewed the roles of endogenous and exogenous SAs in the pathogenesis and treatment of FA. In addition, we considered the structure-function relationship of SAs to provide a theoretical basis for the development of SA-based FA treatments.

Dietary Neu5Ac Intervention Protects Against Atherosclerosis Associated With Human-Like Neu5Gc Loss-Brief Report.

Objective: Species-specific pseudogenization of the CMAH gene during human evolution eliminated common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) biosynthesis from its precursor N-acetylneuraminic acid (Neu5Ac). With metabolic nonhuman Neu5Gc incorporation into endothelia from red meat, the major dietary source, anti-Neu5Gc antibodies appeared. Human-like Ldlr-/-Cmah-/- mice on a high-fat diet supplemented with a Neu5Gc-enriched mucin, to mimic human red meat consumption, suffered increased atherosclerosis if human-like anti-Neu5Gc antibodies were elicited. Approach and Results: We now ask whether interventional Neu5Ac feeding attenuates metabolically incorporated Neu5Gc-mediated inflammatory acceleration of atherogenesis in this Cmah-/-Ldlr-/- model system. Switching to a Neu5Gc-free high-fat diet or adding a 5-fold excess of Collocalia mucoid-derived Neu5Ac in high-fat diet protects against accelerated atherosclerosis. Switching completely from a Neu5Gc-rich to a Neu5Ac-rich diet further reduces severity. Remarkably, feeding Neu5Ac-enriched high-fat diet alone has a substantial intrinsic protective effect against atherosclerosis in Ldlr-/- mice even in the absence of dietary Neu5Gc but only in the human-like Cmah-null background. Conclusions: Interventional Neu5Ac feeding can mitigate or prevent the red meat/Neu5Gc-mediated increased risk for atherosclerosis, and has an intrinsic protective effect, even in the absence of Neu5Gc feeding. These findings suggest that similar interventions should be tried in humans and that Neu5Ac-enriched diets alone should also be investigated further.

Human species-specific loss of CMP-N-acetylneuraminic acid hydroxylase enhances atherosclerosis via intrinsic and extrinsic mechanisms.

Cardiovascular disease (CVD) events due to atherosclerosis cause one-third of worldwide deaths and risk factors include physical inactivity, age, dyslipidemia, hypertension, diabetes, obesity, smoking, and red meat consumption. However, ∼15% of first-time events occur without such factors. In contrast, coronary events are extremely rare even in closely related chimpanzees in captivity, despite human-like CVD-risk-prone blood lipid profiles, hypertension, and mild atherosclerosis. Similarly, red meat-associated enhancement of CVD event risk does not seem to occur in other carnivorous mammals. Thus, heightened CVD risk may be intrinsic to humans, and genetic changes during our evolution need consideration. Humans exhibit a species-specific deficiency of the sialic acid N-glycolylneuraminic acid (Neu5Gc), due to pseudogenization of cytidine monophosphate-N-acetylneuraminic acid (Neu5Ac) hydroxylase (CMAH), which occurred in hominin ancestors ∼2 to 3 Mya. Ldlr-/- mice with human-like Cmah deficiency fed a sialic acids (Sias)-free high-fat diet (HFD) showed ∼1.9-fold increased atherogenesis over Cmah wild-type Ldlr-/- mice, associated with elevated macrophage cytokine expression and enhanced hyperglycemia. Human consumption of Neu5Gc (from red meat) acts as a "xeno-autoantigen" via metabolic incorporation into endogenous glycoconjugates, as interactions with circulating anti-Neu5Gc "xeno-autoantibodies" potentiate chronic inflammation ("xenosialitis"). Cmah-/-Ldlr-/- mice immunized with Neu5Gc-bearing antigens to generate human-like anti-Neu5Gc antibodies suffered a ∼2.4-fold increased atherosclerosis on a Neu5Gc-rich HFD, compared with Neu5Ac-rich or Sias-free HFD. Lesions in Neu5Gc-immunized and Neu5Gc-rich HFD-fed Cmah-/-Ldlr-/- mice were more advanced but unexplained by lipoprotein or glucose changes. Human evolutionary loss of CMAH likely contributes to atherosclerosis predisposition via multiple intrinsic and extrinsic mechanisms, and future studies could consider this more human-like model.

A Deadly Embrace: Hemagglutination Mediated by SARS-CoV-2 Spike Protein at Its 22 N-Glycosylation Sites, Red Blood Cell Surface Sialoglycoproteins, and Antibody.

Rouleaux (stacked clumps) of red blood cells (RBCs) observed in the blood of COVID-19 patients in three studies call attention to the properties of several enveloped virus strains dating back to seminal findings of the 1940s. For COVID-19, key such properties are: (1) SARS-CoV-2 binds to RBCs in vitro and also in the blood of COVID-19 patients; (2) although ACE2 is its target for viral fusion and replication, SARS-CoV-2 initially attaches to sialic acid (SA) terminal moieties on host cell membranes via glycans on its spike protein; (3) certain enveloped viruses express hemagglutinin esterase (HE), an enzyme that releases these glycan-mediated bindings to host cells, which is expressed among betacoronaviruses in the common cold strains but not the virulent strains, SARS-CoV, SARS-CoV-2 and MERS. The arrangement and chemical composition of the glycans at the 22 N-glycosylation sites of SARS-CoV-2 spike protein and those at the sialoglycoprotein coating of RBCs allow exploration of specifics as to how virally induced RBC clumping may form. The in vitro and clinical testing of these possibilities can be sharpened by the incorporation of an existing anti-COVID-19 therapeutic that has been found in silico to competitively bind to multiple glycans on SARS-CoV-2 spike protein.

Comparison of proteins with anti-influenza virus effects in parotid and submandibular-sublingual saliva in humans.

BACKGROUND: Saliva possesses antiviral activity, with submandibular-sublingual (SMSL) saliva having higher antiviral activity than parotid saliva. Various salivary proteins have inactivating effects on influenza A virus (IAV), but the detailed relationship between antiviral proteins and salivary anti-IAV activities in the parotid and SMSL glands is unknown. Here, to identify salivary proteins with anti-IAV activity, salivary proteins from parotid and SMSL glands were identified, quantified, and compared using liquid chromatography-mass spectrometry. METHODS: Twelve healthy male volunteers participated in the study. Parotid and SMSL saliva was collected by suction and collection devices. We assessed anti-IAV activities, protein concentrations, and protein-bound sialic acid concentrations in parotid and SMSL saliva. RESULTS: SMSL had significantly higher anti-IAV activity than parotid saliva. SMSL also had higher concentrations of glycoproteins, such as mucin 5B and mucin 7, protein-bound sialic acid, cystatins, and lysozyme C, compared with parotid saliva. Salivary mucin 5B and mucin 7 concentrations significantly positively correlated with the salivary protein-bound sialic acid concentration. Salivary anti-IAV activity significantly positively correlated with protein-bound sialic acid, mucin 5B, mucin 7, cystatin-C, -S, and -SN concentrations. CONCLUSION: Salivary mucins, cystatins, and lysozyme C contribute to the high anti-IAV activity of SMSL saliva.

Structure and properties of the giant reed (Arundo donax) lectin (ADL).

Arundo donax lectin (ADL) is a 170 amino acid protein that can be purified from the rhizomes of the giant reed or giant cane by exploiting its selective binding to chitin followed by elution with N-acetylglucosamine. The lectin is listed in the UniProt server, the largest protein sequence database, as an uncharacterized protein with chitin-binding domains (A0A0A9P802). This paper reports the purification, structure and ligand-binding properties of ADL. The lectin is a homodimer in which the two protomers are linked by two disulfide bridges. Each polypeptide chain presents four carbohydrate-binding modules that belong to carbohydrate-binding module family 18. A high degree of sequence similarity is observed among the modules present in each protomer. We have determined the X-ray structure of the apo-protein to a resolution of 1.70 Å. The carbohydrate-binding modules, that span a sequence of approximately 40 amino acids, present four internal disulfide bridges, a very short antiparallel central beta sheet and three short alpha helices, two on one side of the beta sheet and one on the other. The structures of the complexes of the lectin with N-acetylglucosamine, N-acetyllactosamine, N-acetylneuraminic acid and N-N'diacetylchitobiose reveal that ADL has two primary and two secondary carbohydrate-binding sites per dimer. They are located at the interface between the two protomers, and each binding site involves residues of both chains. The lectin presents structural similarity to the wheat germ agglutinin family, in particular, to isoform 3.

Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection.

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

Exploring the Effect of Structure-Based Scaffold Hopping on the Inhibition of Coxsackievirus A24v Transduction by Pentavalent N-Acetylneuraminic Acid Conjugates.

Coxsackievirus A24 variant (CVA24v) is the primary causative agent of the highly contagious eye infection designated acute hemorrhagic conjunctivitis (AHC). It is solely responsible for two pandemics and several recurring outbreaks of the disease over the last decades, thus affecting millions of individuals throughout the world. To date, no antiviral agents or vaccines are available for combating this disease, and treatment is mainly supportive. CVA24v utilizes Neu5Ac-containing glycans as attachment receptors facilitating entry into host cells. We have previously reported that pentavalent Neu5Ac conjugates based on a glucose-scaffold inhibit CVA24v infection of human corneal epithelial cells. In this study, we report on the design and synthesis of scaffold-replaced pentavalent Neu5Ac conjugates and their effect on CVA24v cell transduction and the use of cryogenic electron microscopy (cryo-EM) to study the binding of these multivalent conjugates to CVA24v. The results presented here provide insights into the development of Neu5Ac-based inhibitors of CVA24v and, most significantly, the first application of cryo-EM to study the binding of a multivalent ligand to a lectin.

Supplementation With the Sialic Acid Precursor N-Acetyl-D-Mannosamine Breaks the Link Between Obesity and Hypertension.

BACKGROUND: Obesity-related hypertension is a common disorder, and attempts to combat the underlying obesity are often unsuccessful. We previously revealed that mice globally deficient in the inhibitory immunoglobulin G (IgG) receptor FcγRIIB are protected from obesity-induced hypertension. However, how FcγRIIB participates is unknown. Studies were designed to determine if alterations in IgG contribute to the pathogenesis of obesity-induced hypertension. METHODS: Involvement of IgG was studied using IgG µ heavy chain-null mice deficient in mature B cells and by IgG transfer. Participation of FcγRIIB was interrogated in mice with global or endothelial cell-specific deletion of the receptor. Obesity was induced by high-fat diet (HFD), and blood pressure (BP) was measured by radiotelemetry or tail cuff. The relative sialylation of the Fc glycan on mouse IgG, which influences IgG activation of Fc receptors, was evaluated by Sambucus nigra lectin blotting. Effects of IgG on endothelial NO synthase were assessed in human aortic endothelial cells. IgG Fc glycan sialylation was interrogated in 3442 human participants by mass spectrometry, and the relationship between sialylation and BP was evaluated. Effects of normalizing IgG sialylation were determined in HFD-fed mice administered the sialic acid precursor N-acetyl-D-mannosamine (ManNAc). RESULTS: Mice deficient in B cells were protected from obesity-induced hypertension. Compared with IgG from control chow-fed mice, IgG from HFD-fed mice was hyposialylated, and it raised BP when transferred to recipients lacking IgG; the hypertensive response was absent if recipients were FcγRIIB-deficient. Neuraminidase-treated IgG lacking the Fc glycan terminal sialic acid also raised BP. In cultured endothelial cells, via FcγRIIB, IgG from HFD-fed mice and neuraminidase-treated IgG inhibited vascular endothelial growth factor activation of endothelial NO synthase by altering endothelial NO synthase phosphorylation. In humans, obesity was associated with lower IgG sialylation, and systolic BP was inversely related to IgG sialylation. Mice deficient in FcγRIIB in endothelium were protected from obesity-induced hypertension. Furthermore, in HFD-fed mice, ManNAc normalized IgG sialylation and prevented obesity-induced hypertension. CONCLUSIONS: Hyposialylated IgG and FcγRIIB in endothelium are critically involved in obesity-induced hypertension in mice, and supportive evidence was obtained in humans. Interventions targeting these mechanisms, such as ManNAc supplementation, may provide novel means to break the link between obesity and hypertension.

In vivo evolutionary engineering of riboswitch with high-threshold for N-acetylneuraminic acid production.

Riboswitches with desired properties, such as sensitivity, threshold, dynamic range, is important for its application. However, the property change of a natural riboswitch is difficult due to the lack of the understanding of aptamer ligand binding properties and a proper screening method for both rational and irrational design. In this study, an effective method to change the threshold of riboswitch was established in vivo based on growth coupled screening by combining both positive and negative selections. The feasibility of the method was verified by the model library. Using this method, an N-acetylneuraminic acid (NeuAc) riboswitch was evolved and modified riboswitches with high threshold and large dynamic range were obtained. Then, using a new NeuAc riboswitch, both ribosome binding sites and key gene in NeuAc biosynthesis pathway were optimized. The highest NeuAc production of 14.32 g/l that has been reported using glucose as sole carbon source was obtained.

The neuroplasticity marker PSA-NCAM: Insights into new therapeutic avenues for promoting neuroregeneration.

Neuroplastic alterations are the key processes involved in adaptation and rehabilitation after all neurological injuries and pathologies. Being the central contributor to the developmental and adult neuroplasticity, the polysialylated form of Neural Cell Adhesion Molecule (PSA-NCAM) may prove to be a potential target to facilitate repair/regeneration after CNS injury and disease. Over the years, several experimental approaches have been developed to exploit the therapeutic potential of PSA-NCAM. Broadly, the studies focused on cell-transplantation strategies to alter PSA-NCAM properties at the injury site, injection of peptide based as well as synthetic PSA mimetics directly into the injury site or the application of PSA containing hydrogels and scaffolds as biomaterials. A comprehensive understanding of the PSA-based experimental approaches, as well as their pros and cons, is urgently required for successful implementation of this molecule in therapeutics. The current review, therefore, has been designed to give the readers a thorough account of all the diverse roles of PSA in the adult nervous system and the recent progress that has been made in developing PSA-based therapeutic approaches for neuroregeneration.

Correlation of serum N-Acetylneuraminic acid with the risk and prognosis of acute coronary syndrome: a prospective cohort study.

BACKGROUND: N-acetylneuraminic acid (Neu5Ac) is a functional metabolite involved in coronary artery disease (CAD). We aimed to evaluate the relationship between serum Neu5Ac and the risk and prognosis of acute coronary syndrome (ACS) in a real-world prospective study. METHODS: Patients with suspected ACS who underwent coronary angiography were included. Serum Neu5Ac was measured at admission. Coronary lesion severity was evaluated by Gensini Score. GRACE risk stratification was performed at admission. Major adverse cardiac events (MACEs) were recorded during follow-up. RESULTS: A total of 766 patients, including 537 with unstable angina (UAP), 100 with myocardial infarction (MI), and 129 without CAD were included. The circulating Neu5Ac level was significantly higher in patients with MI (median [1QR]: 297[220, 374] ng/ml) than in those with UAP (227 [114, 312] ng/ml) or without CAD (207 [114, 276] ng/ml; both p < 0.001). Serum level of Neu5Ac was positively correlated with age, hypertension, serum uric acid, creatinine, MB isoform of creatine kinase (CK-MB), and Gensini score (all p < 0.05). Receiver operating characteristic curve analysis showed that a higher serum Neu5Ac was potentially associated with MI and high-risk GRACE stratification in ACS patients. Logistic analysis identified only elevated serum Neu5Ac as an independent predictor of MACEs in these patients (odds ratio [OR]: 1.003, 95% confidence interval [CI]: 1.002-1.005, p < 0.001). CONCLUSIONS: Serum Neu5Ac is associated with myocardial injury, GRACE risk category, and prognosis in ACS patients.

A serine protease-associated lectin in the cytolytic system of blowfly (Chrysomya megacephala) larvae: Evidence and characterization.

Cytolytic activity against invading microorganisms is one of the innate forms of immunity in invertebrates. A serine protease-associated sialic acid-specific cytolytic lectin was purified using glutaraldehyde-fixed ox erythrocytes from the larval extract of blowfly (Chrysomya megacephala). The purified lectin lysed vertebrate erythrocytes with effective haemolysis of ox red blood cells (RBCs) in an isotonic medium. The degree of haemolytic (HL) activity of the purified cytolytic lectin depended on its concentration, pH, temperature, and calcium ions. It was sensitive to ethylenediaminetetraacetic acid. The native molecular mass of the C-type lectin was 260 ± 26 kDa, comprising four different polypeptide subunits of 75 kDa (pI ~8), 69 kDa (pI ~7.0), 61 kDa (pI ~5.3), and 55 kDa (pI ~4.6). The association between the C-type lectin and serine protease was confirmed by MALDI-TOF-MS analysis that revealed its homology in the same spectral peak as well as the proteases and phenylmethylsulphonyl fluoride inhibition of HL activity. Haemolysis inhibition by N-acetylneuraminic acid and other sugars revealed the properties of the lectin. The purified lectin distorted the integrity of ox RBCs and Paenalcaligenes hermetiae. This in vitro study documents the presence of a cytolytic system in blowfly (C. megacephala) larvae for the clearance of invading microbial pathogens in their feeding niche.