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.

N-acetylneuraminic acid modulates SQSTM1/p62 sialyation-mediated ubiquitination degradation contributing to vascular endothelium dysfunction in experimental atherosclerosis mice.

Sialic acid (SIA) has been reported to be a risk factor for atherosclerosis (AS) due to its high plasma levels in such patients. However, the effect of increasing SIA in circulation on endothelial function during AS progression remains unclear. In the present study, ApoE-/- mice and endothelial cells line (HUVEC cells) were applied to investigate the effect of SIA on AS progression and its potential molecular mechanism. In vivo, mice were injected intraperitoneally with Neu5Ac (main form of SIA) to keep high-level SIA in circulation. ORO, H&E, and Masson staining were applied to detect the plaque progression. In vitro, HUVECs were treated with Neu5Ac at different times, CCK-8, RT-PCR, western blot, and immunoprecipitation methods were used to analyze its effects on endothelial function and the potential involved mechanism. Results from the present study showed that high plasma levels of Neu5Ac in ApoE-/- mice could aggravate the plaque areas as well as increase necrotic core areas and collagen fiber contents. Remarkably, Neu5Ac levels in circulation displayed a positive correlation with AS plaque areas. Furthermore, results from HUVECs showed that Neu5Ac inhibited cells viability in a time/dose-dependent manner, by then induced the activation of inflammation makers such as ICAM-1 and IL-1ß. Mechanism study showed that the activation of excessive autophagy medicated by SQSTM1/p62 displayed an important role in endothelium inflammatory injury. Neu5Ac could modify SQSTM1/p62 as a sialylation protein, and then increase its level with ubiquitin binding, further inducing ubiquitination degradation and being involved in the excessive autophagy pathway. Inhibition of sialylation by P-3Fax-Neu5Ac, a sialyltransferase inhibitor, reduced the binding of SQSTM1/p62 to ubiquitin. Together, these findings indicated that Neu5Ac increased SQSTM1/p62-ubiquitin binding through sialylation modification, thereby inducing excessive autophagy and subsequent endothelial injury. Inhibition of SQSTM1/p62 sialylation might be a potential strategy for preventing such disease with high levels of Neu5Ac in circulation.

Amphiphilic Sialic Acid Derivatives as Potential Dual-Specific Inhibitors of Influenza Hemagglutinin and Neuraminidase.

In the shadow of SARS-CoV-2, influenza seems to be an innocent virus, although new zoonotic influenza viruses evolved by mutations may lead to severe pandemics. According to WHO, there is an urgent need for better antiviral drugs. Blocking viral hemagglutinin with multivalent N-acetylneuraminic acid derivatives is a promising approach to prevent influenza infection. Moreover, dual inhibition of both hemagglutinin and neuraminidase may result in a more powerful effect. Since both viral glycoproteins can bind to neuraminic acid, we have prepared three series of amphiphilic self-assembling 2-thio-neuraminic acid derivatives constituting aggregates in aqueous medium to take advantage of their multivalent effect. One of the series was prepared by the azide-alkyne click reaction, and the other two by the thio-click reaction to yield neuraminic acid derivatives containing lipophilic tails of different sizes and an enzymatically stable thioglycosidic bond. Two of the three bis-octyl derivatives produced proved to be active against influenza viruses, while all three octyl derivatives bound to hemagglutinin and neuraminidase from H1N1 and H3N2 influenza types.

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC is characterized by a desmoplastic, hypoxic and highly immunosuppressive tumor microenvironment (TME), where T-cell infiltration is often lacking (immune desert), or where T cells are located distant from the tumor islands (immune excluded). Converting the TME to an immune-inflamed state, allowing T-cell infiltration, could increase the success of immunotherapy in PDAC. METHOD: In this study, we use the KPC3 subcutaneous PDAC mouse model to investigate the role of tumor-derived sialic acids in shaping the tumor immune landscape. A sialic acid deficient KPC3 line was generated by genetic knock-out of the CMAS (cytidine monophosphate N-acetylneuraminic acid synthetase) enzyme, a critical enzyme in the synthesis of sialic acid-containing glycans. The effect of sialic acid-deficiency on immunotherapy efficacy was assessed by treatment with anti-programmed cell death protein 1 (PD-1) and agonistic CD40. RESULT: The absence of sialic acids in KPC3 tumors resulted in increased numbers of CD4+ and CD8+ T cells in the TME, and reduced frequencies of CD4+ regulatory T cells (Tregs) within the T-cell population. Importantly, CD8+ T cells were able to infiltrate the tumor islands in sialic acid-deficient tumors. These favorable alterations in the immune landscape sensitized sialic acid-deficient tumors to immunotherapy, which was ineffective in sialic acid-expressing KPC3 tumors. In addition, high expression of sialylation-related genes in human pancreatic cancer correlated with decreased CD8+ T-cell infiltration, increased presence of Tregs, and poorer survival probability. CONCLUSION: Our results demonstrate that tumor-derived sialic acids mediate T-cell exclusion within the PDAC TME, thereby impairing immunotherapy efficacy. Targeting sialic acids represents a potential strategy to enhance T-cell infiltration and improve immunotherapy outcomes in PDAC.

Sialic Acid Enhanced the Antistress Capability under Challenging Situations by Increasing Synaptic Transmission.

BACKGROUND: In early life, sialic acid (SA) plays a crucial role in neurodevelopment and neuronal function. However, it remains unclear whether and how SA supplementation in early life promotes behavioral response to stress in adolescence. OBJECTIVES: This study aimed to examine the effects and mechanisms of SA on the antistress capability under challenging situations. METHODS: In this study, C57BL/6 mice were daily supplemented with 1 µL SA solution/g body weight at the dose of 10 mg/kg/d from postnatal day (PND) 5-45. The antistress behaviors, including open field, elevated plus maze, forced swimming test, and tail suspension test, were performed at PND 46, PND 48, PND 50, and PND 52 to detect the antistress ability of SA, respectively. RESULTS: Our results showed that SA-treated mice were more active in facing challenging situations. The fiber photometry experiment showed that SA promoted the excitatory neuronal response in the medial prefrontal cortex (mPFC), which was extensively interconnected to stress. Besides, electrophysiological results revealed SA enhanced synaptic transmission rather than neuronal excitability of mPFC excitatory neurons. It was also supported by the increasing spine density of mPFC excitatory neurons. At the molecular amount, the SA elevated the transmitter release-related proteins of mPFC, including Synapsin 1 and vesicular glutamate transporter 1 (VGlut 1). Furthermore, SA supplementation enhanced synaptic transmission mainly by altering the kinetics of synaptic transmission. CONCLUSIONS: The SA supplementation enhanced the response capability to stress under challenging situations, and the enhanced synaptic transmission of mPFC excitatory neurons may be the neurological basis of active response under challenging situations. In general, our findings suggested that SA supplementation in early life can promote stress resistance in adolescence.

Cannabidiol and Cannabigerol Modify the Composition and Physicochemical Properties of Keratinocyte Membranes Exposed to UVA.

The action of UVA radiation (both that derived from solar radiation and that used in the treatment of skin diseases) modifies the function and composition of keratinocyte membranes. Therefore, this study aimed to assess the effects of phytocannabinoids (CBD and CBG), used singly and in combination, on the contents of phospholipids, ceramides, lipid rafts and sialic acid in keratinocyte membranes exposed to UVA radiation, together with their structure and functionality. The phytocannabinoids, especially in combination (CBD+CBG), partially prevented increased levels of phosphatidylinositols and sialic acid from occurring and sphingomyelinase activity after the UVA exposure of keratinocytes. This was accompanied by a reduction in the formation of lipid rafts and malondialdehyde, which correlated with the parameters responsible for the integrity and functionality of the keratinocyte membrane (membrane fluidity and permeability and the activity of transmembrane transporters), compared to UVA-irradiated cells. This suggests that the simultaneous use of two phytocannabinoids may have a protective effect on healthy cells, without significantly reducing the therapeutic effect of UV radiation used to treat skin diseases such as psoriasis.

Transforms of Cell Surface Glycoproteins Charge Influences Tumor Cell Metastasis via Atypically Inhibiting Epithelial-Mesenchymal Transition Including Matrix Metalloproteinases and Cell Junctions.

Cell communication and signal transduction rely heavily on the charge on the cell surface. The cell surface is negatively charged, with glycoproteins on the cell membrane providing a large percentage of the charge. Sialic acid is found on the outermost side of glycan chains and contributes to glycoprotein's negative charge. Sialic acid is highly expressed in tumor cells and plays an important role in tumor metastasis and immune escape by interacting with extracellular ligands. However, the specific effect of negative charge changes on glycoproteins is still poorly understood. In this study, we used 9-azido sialic acid (9Az-Sia) to create artificial epitopes on glycoproteins via metabolic glycan labeling, and we attached charged groups such as amino and carboxyl to 9Az-Sia via a click reaction with dibenzocyclooctyne (DBCO). The charge of glycoproteins was changed by metabolic glycan labeling and click modification. The results showed that the migration and invasion ability of the MDA-MB-231 cell labeled with 9Az-Sia was significantly reduced after the modification with amino groups rather than carboxyl groups. Epithelial-mesenchymal transition (EMT) is the biological process of metastatic tumor cells, with an increasing ability of tumor cells to migrate and invade. In particular, the expression of adhesion molecules increased in the amine-linked group, whereas the expression of matrix metalloproteinases (MMPs) increased significantly, which is not identical to EMT characteristics. In vivo experiments have demonstrated that the loss of negative charge on glycoproteins has an inhibitory effect on tumors. In conclusion, modifying the positive charge on the surface of glycoproteins can inhibit tumor cell metastasis and has great potential for tumor therapy.

Antiobesity Effect of N-Acetylneuraminic Acid by Enhancing Antioxidative Capacity in Mice Fed a High-Fat Diet.

The sialic acid N-acetylneuraminic acid (NANA), an essential factor in bioregulation, is a functional food component that is known to have beneficial health effects, but its antiobesity effect has not been clearly understood. Adipocyte dysfunction in obesity involves a decrease in the level of NANA sialylation. In this study, we investigated the antiobesity effect of NANA in mice fed a high-fat diet (HFD) and in 3T3-L1 adipocytes. Male C57BL/6J mice were randomly divided into three groups and administered the following diets: a normal diet, an HFD, and an HFD with 1% NANA supplementation for 12 weeks. NANA supplementation significantly reduced body weight gain; epididymal adipose tissue hypertrophy; and serum lipid, fasting glucose, and aspartate transaminase levels compared with those in HFD mice. The percentage of lipid droplets in hepatic tissue was also decreased by NANA supplementation in HFD mice. The downregulation of Adipoq expression and upregulation of Fabp4 expression induced by HFD in epididymal adipocytes were improved by NANA supplementation. The downregulation of Sod1 expression and increase in malondialdehyde level were induced by HFD, and they were significantly improved in the liver by NANA supplementation, but not in epididymal adipocytes. However, NANA supplementation had no effect on sialylation and antioxidant enzyme levels in mouse epididymal adipocytes and 3T3-L1 adipocytes. Overall, NANA exerts antiobesity and antihypolipidemic effects and may be beneficial in suppressing obesity-related diseases.

Increased α-2,6 sialic acid on microglia in amyloid pathology is resistant to oseltamivir.

Terminal sialic acid residues are present on most glycoproteins and glycolipids, but levels of sialylation are known to change in the brain throughout the lifespan as well as during disease. Sialic acids are important for numerous cellular processes including cell adhesion, neurodevelopment, and immune regulation as well as pathogen invasion into host cells. Neuraminidase enzymes, also known as sialidases, are responsible for removal of terminal sialic acids in a process known as desialylation. Neuraminidase 1 (Neu1) cleaves the α-2,6 bond of terminal sialic acids. Aging individuals with dementia are often treated with the antiviral medication oseltamivir, which is associated with induction of adverse neuropsychiatric side effects; this drug inhibits both viral and mammalian Neu1. The present study tested whether a clinically relevant antiviral dosing regimen of oseltamivir would disrupt behavior in the 5XFAD mouse model of Alzheimer's disease amyloid pathology or wild-type littermates. While oseltamivir treatment did not impact mouse behavior or modify amyloid plaque size or morphology, a novel spatial distribution of α-2,6 sialic acid residues was discovered in 5XFAD mice that was not present in wild-type littermates. Further analyses revealed that α-2,6 sialic acid residues were not localized the amyloid plaques but instead localized to plaque-associated microglia. Notably, treatment with oseltamivir did not alter α-2,6 sialic acid distribution on plaque-associated microglia in 5XFAD mice which may be due to downregulation of Neu1 transcript levels in 5XFAD mice. Overall, this study suggests that plaque-associated microglia are highly sialylated and are resistant to change with oseltamivir, thus interfering with microglia immune recognition of and response to amyloid pathology.

Design, Synthesis, and Antiviral Evaluation of Sialic Acid Derivatives as Inhibitors of Newcastle Disease Virus Hemagglutinin-Neuraminidase: A Translational Study on Human Parainfluenza Viruses.

Global infections with viruses belonging to the Paramyxoviridae, such as Newcastle disease virus (NDV) or human parainfluenza viruses (hPIVs), pose a serious threat to animal and human health. NDV-HN and hPIVs-HN (HN hemagglutinin-neuraminidase) share a high degree of similarity in catalytic site structures; therefore, the development of an efficient experimental NDV host model (chicken) may be informative for evaluating the efficacy of hPIVs-HN inhibitors. As part of the broad research in pursuit of this goal and as an extension of our published work on antiviral drug development, we report here the biological results obtained with some newly synthesized C4- and C5-substituted 2,3-unsaturated sialic acid derivatives against NDV. All developed compounds showed high neuraminidase inhibitory activity (IC50 0.03-13 µM). Four molecules (9, 10, 23, 24) confirmed their high in vitro inhibitory activity, which caused a significant reduction of NDV infection in Vero cells, accompanied by very low toxicity.

Targeting of TAMs with freeze-dried monosialotetrahexosylganglioside and sialic acid-octadecylamine co-modified liposomes remodels the tumor microenvironment and enhances anti-tumor activity.

Although anti-tumor strategies targeting tumor-associated immune cells were being rapidly developed, the preparations were usually limited in targeting efficiency. To overcome this barrier, this study reported a novel sialic acid-octadecylamine (SA-ODA) and monosialotetrahexosylganglioside (GM1) co-modified epirubicin liposomes (5-5-SAGL-EPI), which improved tumor-targeting ability through the active targeting of tumor-associated macrophages (TAMs) by SA-ODA and the long circulation of GM1. Thus, we evaluated 5-5-SAGL-EPI in vitro and in vivo. Analysis of cellular uptake by RAW264.7 cells using flow cytometry and confocal microscopy showed a higher rate of cellular uptake for 5-5-SAGL-EPI than for the common liposomes (CL-EPI). In pharmacokinetic studies using Wistar rats, compared to CL-EPI, 5-5-SAGL-EPI showed a higher circulation time in vivo. Tissue distribution studies in Kunming mice bearing S180 tumors revealed increased distribution of 5-5-SAGL-EPI in tumor tissues compared with liposomes modified with single ligands (SA-ODA [5-SAL-EPI] or GM1 [5-GL-EPI]). In vivo anti-tumor experiments using the S180 tumor-bearing mice revealed a high tumor inhibition rate and low toxicity for 5-5-SAGL-EPI. Moreover, freeze-dried 5-5-SAGL-EPI had good storage stability, and the anti-tumor effect was comparable to that before freeze-drying. Overall, 5-5-SAGL-EPI exhibited excellent anti-tumor effects before and after lyophilization.

Short term deuterium depletion in drinking water reduced tumor induced oxidative stress in mice liver.

The aim of current work was able to show the oxidant effect of cancer cells found in any part of the body on the liver and to investigate the possible protective effect of deuterium-depleted water (DDW) on this oxidant effect by determining of some liver parameters. Ehrlich ascites tumor bearing BALB/c mice were used for this purpose. BALB/c mice were selected randomly and divided into four groups (n = 5 in each group) as control group, tumor group, control+DDW group, tumor+DDW group, fifteen days after tumor cell injection, liver tissue samples were taken for all groups. In the tumor group, liver lipid peroxidation, sialic acid and protein carbonyl levels, xanthine oxidase, myeloperoxidase, catalase, gamma-glutamyl transferase, sorbitol dehydrogenase, glutathione peroxidase and glutathione reductase activities, were significantly higher than those in the control group while glutathione levels and paraoxonase1, sodium potassium ATPase, glutathione-S-transferase, alanine transaminase and aspartate transaminase activities decreased significantly. Compared with the tumor group, the changes in all parameters except sialic acid, catalase, alanine transaminase and aspartate transaminase were reversed in the DDW given tumor groups, while sialic acid and catalase values continued to increase, and alanine transaminase and aspartate transaminase values continued to decrease. In conclusion, the consumption of DDW may be beneficial and protective against excessive oxidative stress in cancer complications.

[Basic Research on Bullfrog Egg-derived Sialic Acid-binding Lectin for Cancer Treatment].

Sialic acid-binding lectin from Rana catesbeiana (cSBL) is a multifunctional protein with both lectin and ribonuclease activity and is, therefore, called a leczyme. It exerts cancer cell-selective antitumor effects on a variety of cancer cells in vitro and in vivo under conditions where no undesired side effects are observed. cSBL elicits antitumor effects by degrading cellular RNA and subsequently inducing apoptosis via a pathway mediated by mitochondria and endoplasmic reticulum stress. Further, it exerts synergistic antitumor effects with other molecules such as tumor necrosis factor-related apoptosis-inducing ligand and pemetrexed. Recent studies have revealed that long-term treatment of cancer cells with cSBL causes significant pleiotropic changes in the expression profiles of several genes, including multiple genes involved in metabolic pathways. Furthermore, cSBL reduces the expression of some cancer-related molecules such as human epidermal growth factor receptors, aldo-keto reductase 1B10, and ATP-binding cassette transporter C2. The information described above is expected to lead to useful applications, such as effective regimens comprising cSBL and other drugs. These findings reveal favorable properties of cSBL as an anticancer drug, which may contribute to the development of new therapeutic strategies for cancer treatment.

Optimization design of sialic acid derivatives enhances the performance of liposomes for modulating immunosuppressive tumor microenvironments.

AIMS: Sialic acid derivatives (SA-derivatives) provide a nanomedicine platform for tumor-targeted delivery and treatment, and allow modulation of immunosuppressive tumor microenvironments with excellent therapeutic effects. Further, the multi-reactive groups of sialic acid (SA) contribute to the diversity of SA derivatives, which inevitably has implications for drug delivery systems and tumor therapy. However, relevant research remains lacking at present. Therefore, this study aimed to explore the effects of SA derivatives on SA-mediated drug delivery systems. MAIN METHODS: Four SA-derivatives with different linking bonds (ester and amide bonds), different linking groups (hydroxyl and carboxyl), and different linking objects (cholesterol, octadecanoic acid, and octadecylamine) were synthesized and the respective SA derivative-modified doxorubicin liposomes were prepared. In-depth research was conducted using both cells and animals. KEY FINDINGS: We found that an SA-cholesterol conjugate (SA-CH; linking bond, amide bond; linking group, carboxyl; linking object, cholesterol) could improve liposome stability, reduce liposome adsorption to plasma proteins, and enhance the targeting of liposomes for killing tumor-associated macrophages (TAMs). Reduced TAMs in the immunosuppressive tumor microenvironment lead to enhanced tumor infiltration of CD8+ T cells. SIGNIFICANCE: The results of this experiment provide clarity for research and development on SA-derivatives and a theoretical basis for clinical trials of SA-derivative-modified nanoparticles.

Synthesis and Neuraminidase Inhibitory Activity of Sialic Acid Analogues with Fluoro, Phosphono, and Sulfo Functionalities.

Methods to synthesize influenza virus inhibitors with fluoro, phosphono, and/or sulfo functional groups are described. The resulting sialic acid analogues are produced from the natural substrate N-acetylneuraminic acid as starting material. Fluorescent assay methods for inhibition of influenza neuraminidase and virus proliferation are also provided.

Gastroprotective effect of vitamin U in D-galactosamine-induced hepatotoxicity.

Galactosamine (GalN) is a well-known agent for inducing viral hepatitis models in rodents, but it can cause toxicity on different organs. Vitamin U (Vit U) has been proved as a powerful antioxidant on many toxicity models. The present study was designed to investigate the protective effects of Vit U on GalN-induced stomach injury. Rats were divided into four groups as follows: control (group I), Vit U given animals (50 mg/kg per day; group II), GalN administered animals (500 mg/kg at a single dose; group III), GalN + Vit U given animals (at the same dose and time, group IV). At the end of the 3rd day, animals were killed, and stomach tissues were taken. They were homogenized and centrifuged. In comparison to the control group, glutathione, total antioxidant capacity levels, catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, and Na+ /K+ -ATPase activities of GalN group were found to be decreased. On the contrary, lipid peroxidation, advanced oxidized protein products, hexose-hexosamine, fucose, sialic acid, reactive oxygen species levels, as well as the activities of myeloperoxidase, xanthine oxidase, and lactate dehydrogenase were elevated. Administration of Vit U reversed these abnormalities in the GalN group. It can be concluded that Vit U exerts its unique antioxidant effect and prevents GalN-induced gastric damage.

Design and Functional Analysis of Heterobifunctional Multivalent Phage Capsid Inhibitors Blocking the Entry of Influenza Virus.

Multiple conjugation of virus-binding ligands to multivalent carriers is a prominent strategy to construct highly affine virus binders for the inhibition of viral entry into host cells. In a previous study, we introduced rationally designed sialic acid conjugates of bacteriophages (Qß) that match the triangular binding site geometry on hemagglutinin spike proteins of influenza A virions, resulting in effective infection inhibition in vitro and in vivo. In this work, we demonstrate that even partially sialylated Qß conjugates retain the inhibitory effect despite reduced activity. These observations not only support the importance of trivalent binding events in preserving high affinity, as supported by computational modeling, but also allow us to construct heterobifunctional modalities. Capsids carrying two different sialic acid ligand-linker structures showed higher viral inhibition than their monofunctional counterparts. Furthermore, capsids carrying a fluorescent dye in addition to sialic acid ligands were used to track their interaction with cells. These findings support exploring broader applications as multivalent inhibitors in the future.

Sialic Acid Derivatives Inhibit SiaT Transporters and Delay Bacterial Growth.

Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant Staphylococcus aureus. This study represents the first example and a promising lead in developing sialic acid uptake inhibitors as novel antibacterial agents.

N-Acetyl Neuraminic Acid (NANA) Activates L-Type Calcium Channels on Isolated Tentacle Supporting Cells of the Sea Anemone (Aiptasia pallida).

AbstractSensory receptors control nematocyst discharge on sea anemone tentacles. Micromolar N-acetylated sugars (e.g., N-acetyl neuraminic acid [NANA]) bind chemoreceptors on ectodermal supporting cells and predispose adjacent nematocyst discharge in response to mechanical contact via a cyclic adenosine monophosphate (cAMP)-dependent sensitization pathway, while higher NANA levels dose-dependently desensitize. Recent evidence implicates L-type calcium channels in desensitizing the pathway in aconitate sea anemones Aiptasia pallida (also known as Exaiptasia diaphana). We, therefore, hypothesize that NANA activates calcium influx via L-type calcium channels. We demonstrate a dose-dependent, NANA-activated 45Ca influx into dissociated ectodermal cells isolated from A. pallida tentacles, with maximal influx occurring at desensitizing concentrations of NANA. The L-type calcium channel inhibitors nifedipine, diltiazem, methoxyverapamil, and cadmium blocked NANA-stimulated 45Ca influx. Elevated extracellular KCl levels dose-dependently increased nifedipine-sensitive 45Ca influx to implicate voltage-gated calcium channels. Forskolin, 8-bromo-cAMP, and the protein kinase A inhibitor H-8 affect NANA-stimulated calcium influx in a manner consistent with activated cAMP-dependent pathway involvement. Because NANA chemoreceptors localize to supporting cells of cnidocyte supporting cell complexes, NANA activation of 45Ca influx into isolated tentacle ectodermal cells suggests that L-type calcium channels and NANA chemoreceptors co-localize to supporting cells. Indeed, a fluorescent marker of L-type calcium channels localizes to the apical ectoderm adjacent to nematocysts of live tentacles. We conclude that supporting cell chemoreceptors activate co-localized L-type calcium channels via a cAMP-dependent mechanism in order to initiate desensitization. We suggest that pathway desensitization may conserve nematocysts from excessive discharge during prey capture.

Combination immunotherapy of chlorogenic acid liposomes modified with sialic acid and PD-1 blockers effectively enhances the anti-tumor immune response and therapeutic effects.

Melanoma is one of the most common malignant tumors. The anti-PD-1 antibody is used for the treatment of metastatic melanoma. Treatment success is only 35-40% and a range of immune-related adverse reactions can occur. Combination of anti-PD1 antibody therapy with other oncology therapies has been attempted. Herein, we assessed whether chlorogenic acid liposomes modified with sialic acid (CA-SAL) combined with anti-PD1 antibody treatment was efficacious as immunotherapy for melanoma. CA-SAL liposomes were prepared and characterized. In a mouse model of B16F10 tumor, mice were treated with an anti-PD1 antibody, CA-SAL, or combination of CA-SAL + anti-PD1 antibody, and compared with no treatment controls. The tumor inhibition rate, tumor-associated macrophages (TAMs) phenotype, T-cell activity, and safety were investigated. We observed a significant decrease in the proportion of M2-TAMs and CD4+Fop3+ T cells, while there was a significant increase in the proportion of M1-TAMs and CD8+ T cells, and in the activity of T cells, and thus in the tumor inhibition rate. No significant toxicity was observed in major organs. CA-SAL and anti-PD1 Ab combination therapy presented synergistic anti-tumor activity, which enhanced the efficacy of the PD-1 checkpoint blocker in a mouse model of melanoma. In summary, combination immunotherapy of CA-SAL and anti-PD1 Ab has broad prospects in improving the therapeutic effect of melanoma, and may provide a new strategy for clinical treatment.