RESUMO
Influenza viruses remain a major threat to human health. Four classes of drugs have been approved for the prevention and treatment of influenza infections. Oseltamivir, a neuraminidase inhibitor, is a first-line anti-influenza drug, and baloxavir is part of the newest generation of anti-influenza drugs that targets the viral polymerase. The emergence of drug resistance has reduced the efficacy of established antiviral drugs. Combination therapy is one of the options for controlling drug resistance and enhancing therapeutical efficacies. Here, we evaluate the antiviral effects of baloxavir combined with neuraminidase inhibitors (NAIs) against wild-type influenza viruses, as well as influenza viruses with drug-resistance mutations. The combination of baloxavir with NAIs led to significant synergistic effects; however, the combination of baloxavir with laninamivir failed to result in a synergistic effect on influenza B viruses. Considering the rapid emergence of drug resistance to baloxavir, we believe that these results will be beneficial for combined drug use against influenza.
Assuntos
Antivirais , Dibenzotiepinas , Farmacorresistência Viral , Sinergismo Farmacológico , Inibidores Enzimáticos , Morfolinas , Neuraminidase , Piridonas , Triazinas , Dibenzotiepinas/farmacologia , Antivirais/farmacologia , Triazinas/farmacologia , Morfolinas/farmacologia , Piridonas/farmacologia , Neuraminidase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Humanos , Vírus da Influenza B/efeitos dos fármacos , Animais , Piridinas/farmacologia , Tiazóis/farmacologia , Guanidinas/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Cães , Células Madin Darby de Rim Canino , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Ácidos Siálicos , Vírus da Influenza A/efeitos dos fármacos , Tiepinas/farmacologia , Triazóis/farmacologia , Benzimidazóis/farmacologia , PiranosRESUMO
Influenza A and influenza B viruses (FLUAV and FLUBV, respectively) cause significant respiratory disease, hospitalization, and mortality each year. Despite causing at least 25% of the annual disease burden, FLUBV is historically understudied. Unlike FLUAVs, which possess pandemic potential due to their many subtypes and broad host range, FLUBVs are thought to be restricted to only humans and are limited to two lineages. The hemagglutinins (HA) of both influenza types bind glycans terminating in α2,6- or α2,3-sialic acids. For FLUAV, the tropism of human- and avian-origin viruses is well-defined and determined by the terminal sialic acid configuration the HA can accommodate, with avian-origin viruses binding α2,3-linked sialic acids and human-origin viruses binding α2,6-linked sialic acids. In contrast, less is known about FLUBV receptor binding and its impact on host tropism. This review discusses the current literature on FLUBV receptor specificity, HA glycosylation, and their roles in virus tropism, evolution, and infection. While the focus is on findings in the past dozen years, it should be noted that the most current approaches for measuring virus-glycan interactions have not yet been applied to FLUBV and knowledge gaps remain.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza B , Influenza Humana , Receptores Virais , Tropismo Viral , Humanos , Receptores Virais/metabolismo , Animais , Vírus da Influenza B/fisiologia , Vírus da Influenza B/metabolismo , Influenza Humana/virologia , Influenza Humana/metabolismo , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Glicosilação , Ligação Viral , Vírus da Influenza A/metabolismo , Vírus da Influenza A/fisiologia , Ácidos Siálicos/metabolismo , Aves/virologia , Especificidade de HospedeiroRESUMO
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).
Assuntos
Clostridium botulinum , Flagelina , Glicosiltransferases , Especificidade por Substrato , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/química , Flagelina/metabolismo , Flagelina/genética , Flagelina/química , Clostridium botulinum/enzimologia , Clostridium botulinum/metabolismo , Clostridium botulinum/genética , Glicosilação , Escherichia coli/genética , Escherichia coli/metabolismo , Açúcares Ácidos/metabolismo , Engenharia de Proteínas , Ácido N-Acetilneuramínico/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Ácidos SiálicosRESUMO
The presence and the level of antibodies in human sera against bacterial glycans are indications of prior encounters with similar antigens and/or the bacteria that express them by the immune system. An increasing number of pathogenic bacteria that cause human diseases have been shown to express polysaccharides containing a bacterial nonulosonic acid called 5,7-di-N-acetyllegionaminic acid (Leg5,7Ac2). To investigate the immune recognition of Leg5,7Ac2, which is critical for the fight against bacterial infections, a highly effective chemoenzymatic synthon strategy was applied to construct a library of α2-3/6-linked Leg5,7Ac2-glycans via their diazido-derivatives (Leg5,7diN3-glycans) formed by efficient one-pot three-enzyme (OP3E) synthetic systems from a diazido-derivative of a six-carbon monosaccharide precursor. Glycan microarray studies using this synthetic library of a Leg5,7Ac2-capped collection of diverse underlying glycan carriers and their matched sialoside counterparts revealed specific recognition of Leg5,7Ac2 by human IgG antibodies pooled from thousands of healthy donors (IVIG), suggesting prior human encounters with Leg5,7Ac2-expressing pathogenic bacteria at the population level. These biologically relevant Leg5,7Ac2-glycans and their immune recognition assays are important tools to begin elucidating their biological roles, particularly in the context of infection and host-pathogen interactions.
Assuntos
Imunoglobulina G , Análise em Microsséries , Polissacarídeos , Ácidos Siálicos , Humanos , Polissacarídeos/imunologia , Polissacarídeos/química , Imunoglobulina G/imunologia , Análise em Microsséries/métodos , Ácidos Siálicos/química , Açúcares Ácidos/química , Açúcares Ácidos/metabolismo , Anticorpos Antibacterianos/imunologiaRESUMO
Here we report an enzymatic approach to synthesize N-formylneuraminic acid (Neu5Fo) containing sialosides, through a five-enzyme cascade. This method stands as an alternative to traditional chemical syntheses, aiming for precision and efficiency in generating sialosides with a tailored N-formyl group generated directly from formic acid. The newly synthesized Neu5Fo was characterized using various NMR techniques revealing a conformational equilibrium at the amide bond of the formyl group in slow exchange on the NMR time scale with a trans : cis ratio of â¼2 : 1. This work not only suggests potential for exploring the biological roles of sialosides but also points to the possibility of developing novel therapeutic agents.
Assuntos
Ácidos Siálicos , Ácidos Siálicos/química , Ácidos Siálicos/síntese química , Ácidos Siálicos/metabolismo , Formiatos/química , Formiatos/síntese química , Formiatos/metabolismoRESUMO
Fludarabine (FA) is still considered as a first-line chemotherapy drug for hematological tumors related to B lymphocytes. However, it is worth noting that the non-specific distribution and non-different cytotoxicity of FA may lead to irreversible consequences such as central nervous system damage such as blindness, coma, and even death. Therefore, it is very important to develop a system to targeting delivery FA. In preliminary studies, it was found that B lymphoma cells would specific highly expressing the sialic acid-binding immunoglobulin-like lectin 2 (known as CD22). Inspired by the specific recognition of sialic acid residues and CD22, we have developed a supramolecular prodrug based on polysialic acid, an endogenous biomacromolecule, achieving targeted-therapy of B-cell non-Hodgkin's lymphoma (B-NHL). Specifically, the prepared hydrophobic reactive oxygen species-responsive FA dimeric prodrug (F2A) interacts with the TPSA, which polysialic acid were modified by the thymidine derivatives, through non-covalent intermolecular interactions similar to "Watson-Crick" base pairing, resulting in the formation of nanoscale supramolecular prodrug (F@TPSA). Cell experiments have confirmed that F@TPSA can be endocytosed by CD22+ B lymphoma cells including Raji and Ramos cells, and there is a significant difference of endocytosis in other leukocytes. Furthermore, in B-NHL mouse model, compared with FA, F@TPSA is determined to have a stronger tumor targeting and inhibitory effect. More importantly, the distribution of F@TPSA in vivo tends to be enriched in lymphoma tissue rather than nonspecific, thus reducing the leukopenia of FA. The targeted delivery system based on PSA provides a new prodrug modification strategy for targeted treatment of B-NHL.
Assuntos
Linfoma de Células B , Pró-Fármacos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Animais , Camundongos , Humanos , Linhagem Celular Tumoral , Linfoma de Células B/tratamento farmacológico , Ácidos Siálicos/química , Ácidos Siálicos/farmacologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Antineoplásicos/farmacologia , Antineoplásicos/química , Nanopartículas/química , Medicina de Precisão/métodos , Sistemas de Liberação de Medicamentos/métodos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Linfoma não Hodgkin/tratamento farmacológicoRESUMO
Amyloid beta (Aß) aggregation is one of the distinctive pathological hallmarks of Alzheimer's disease (AD). Therefore, the development of effective inhibitors against Aß aggregate formation offers great promise for the treatment of AD. In this study, we designed a novel negatively charged functionalized conjugate aimed at inhibiting Aß42 aggregation and attenuating neurotoxicity by grafting polysialic acid with mannuronate oligosaccharide, a biocompatible glycan extracted from seaweeds, designated as polysialic acid-mannan conjugate (PSA-MOS). ThT, biological microscopy, TEM and CD confirmed the inhibition of Aß42 aggregation by PSA-MOS, as well as its ability to inhibit the conformational transition of Aß42 to ß-sheet. CCK-8 assay demonstrated that PSA-MOS was not cytotoxic to SH-SY5Y (p < 0.05) and promoted cell proliferation. In the Aß42-induced SH-SY5Y injury models, PSA-MOS dose-dependently ameliorated cytotoxicity (p < 0.0001) and significantly reduced the levels of inflammatory factors of IL-1ß (p < 0.0001), IL-6 (p < 0.0001) and TNF-α (p < 0.05). MD simulations demonstrated that PSA-MOS effectively impeded the α-helix to ß-sheet transition of the Aß42 monomer via electrostatic interactions with its CTR and NTR regions. These findings demonstrate the therapeutic potential of PSA-MOS as promising glycoconjugate for the treatment of AD.
Assuntos
Peptídeos beta-Amiloides , Inflamação , Ácidos Siálicos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/química , Humanos , Ácidos Siálicos/química , Ácidos Siálicos/farmacologia , Inflamação/tratamento farmacológico , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Linhagem Celular Tumoral , Mananas/farmacologia , Mananas/química , Proliferação de Células/efeitos dos fármacos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Simulação de Dinâmica Molecular , Ácidos HexurônicosRESUMO
Cancer exploits different mechanisms to escape T-cell immunosurveillance, including overexpression of checkpoint ligands, secretion of immunosuppressive molecules, and aberrant glycosylation. Herein, we report that IFNγ, a potent immunomodulator secreted in the tumor microenvironment, can induce α2,6 hypersialylation in cancer cell lines derived from various histologies. We focused on Siglec-9, a receptor for sialic acid moieties, and demonstrated that the Siglec-9+ T-cell population displayed reduced effector function. We speculated that Siglec-9 in primary human T cells can act as a checkpoint molecule and demonstrated that knocking out Siglec-9 using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system enhanced the functionality of primary human T cells. Finally, we aimed to augment cancer-specific T-cell activity by taking advantage of tumor hypersialylation. Thus, we designed several Siglec-9-based chimeric switch receptors (CSR), which included an intracellular moiety derived from costimulatory molecules (CD28/41BB) and different hinge regions. In an antigen-specific context, T cells transduced with Siglec-9 CSRs demonstrated increased cytokine secretions and upregulation of activation markers. Moreover, T cells equipped with specific Siglec-9 CSRs mediated robust antitumor activity in a xenograft model of human tumors. Overall, this work sheds light on tumor evasion mechanisms mediated by sialylated residues and exemplifies an approach to improve engineered T cell-based cancer treatment. See related Spotlight by Abken, p. 1310.
Assuntos
Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Linfócitos T , Humanos , Animais , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ácidos Siálicos/metabolismo , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/genética , Sistemas CRISPR-Cas , Microambiente Tumoral/imunologiaRESUMO
Highly pathogenic H5N1 avian influenza (HPAI H5N1) viruses occasionally infect, but typically do not transmit, in mammals. In the spring of 2024, an unprecedented outbreak of HPAI H5N1 in bovine herds occurred in the USA, with virus spread within and between herds, infections in poultry and cats, and spillover into humans, collectively indicating an increased public health risk1-4. Here we characterize an HPAI H5N1 virus isolated from infected cow milk in mice and ferrets. Like other HPAI H5N1 viruses, the bovine H5N1 virus spread systemically, including to the mammary glands of both species, however, this tropism was also observed for an older HPAI H5N1 virus isolate. Bovine HPAI H5N1 virus bound to sialic acids expressed in human upper airways and inefficiently transmitted to exposed ferrets (one of four exposed ferrets seroconverted without virus detection). Bovine HPAI H5N1 virus thus possesses features that may facilitate infection and transmission in mammals.
Assuntos
Furões , Virus da Influenza A Subtipo H5N1 , Infecções por Orthomyxoviridae , Ácidos Siálicos , Animais , Furões/virologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Virus da Influenza A Subtipo H5N1/fisiologia , Bovinos , Camundongos , Humanos , Infecções por Orthomyxoviridae/virologia , Infecções por Orthomyxoviridae/transmissão , Feminino , Ácidos Siálicos/metabolismo , Masculino , Leite/virologia , Influenza Humana/transmissão , Influenza Humana/virologia , Influenza Humana/epidemiologia , Glândulas Mamárias Animais/virologia , Virulência , Tropismo Viral , Camundongos Endogâmicos BALB CRESUMO
Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas-deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas-deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells.
Assuntos
Camundongos Knockout , Ácidos Siálicos , Linfócitos T , Animais , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ácidos Siálicos/metabolismo , Sobrevivência Celular , Camundongos Endogâmicos C57BL , Apoptose , Complemento C3/metabolismo , Complemento C3/imunologia , Complemento C3/genética , Oxigenases de Função MistaRESUMO
BACKGROUND: In sharp contrast with analysis of N-glycan that can be prepared by PNGase F, O-glycan analysis remains challenging due to a lack of versatile and simple procedures, especially those mediating cleavage of O-glycans from proteins. Most N-glycans and O-glycans are modified with sialic acids at the non-reducing end and their glycosidic linkages are labile, making it difficult to measure glycans by mass spectrometric analysis. In addition, sialic acid residues present on glycan chains via α2,3-, α2,6-, and α2,8-linkages as structural isomers. RESULTS: In this study, we firstly established a direct and linkage-specific derivatization method for sialylated O-glycans on proteins via linkage-specific lactone-opening aminolysis. In this procedure, labile sialylated glycans were not only stabilized, but also allowed distinguishing between sialyl linkages. Furthermore, we revealed that general reductive ß-elimination was not useful for O-glycan cleavages with undesirable degradations of resulting methyl amides. Using ß-elimination in the presence of pyrazolone (PMP), with low pH despite alkali base concentration, SALSA-derivatized O-glycans could be cleaved with minimal degradations. Cleaved and PMP-labeled O-glycans could be efficiently prepared in an open reaction system at high temperature (evaporative BEP reaction) and detected by simple liquid-phase extraction. Moreover, in the evaporative BEP reaction by changing the alkali solution with LiOH, the lithiated O-glycans could be observed and provided a lot of fragment information reflecting the complex structure of the O-glycans. SIGNIFICANCE: Direct sialic acid linkage-specific derivatization of O-glycans on glycoproteins is simple protocol containing in-solution aminolysis-SALSA and acetonitrile precipitation for removal of excess reagents. Evaporative ß-elimination with pyrazolone makes possible intact O-linked glycan analysis just by liquid-phase extraction. These analytical methods established by the appropriate combination of direct-SALSA and evaporative ß-elimination will facilitate O-glycomic studies in various biological samples.
Assuntos
Polissacarídeos , Ácidos Siálicos , Polissacarídeos/química , Ácidos Siálicos/químicaRESUMO
Living acute brain slices provide a practical platform for imaging sialylation in human brain pathology. However, the limited lifespan of acute brain slices has impeded the use of metabolic glycan labeling (MGL), which requires long-term incubation of clickable unnatural sugars such as N-azidoacetylmannosamine (ManNAz) to metabolically incorporate azides into sialoglycans. Here, we report a fast variant of MGL (fMGL), in which ManNAz-6-phosphate enables efficient azidosugar incorporation within 12 h by bypassing the bottleneck step in the sialic acid biosynthesis pathway, followed by click-labeling with fluorophores and imaging of sialoglycans in acute brain slices from mice and human patients. In the clinical samples of ganglioglioma, fMGL-based imaging reveals specific upregulation of sialylation in astrocyte-like but not neuron-like tumor cells. In addition, fMGL is integrated with click-expansion microscopy for high-resolution imaging of sialoglycans in brain slices. The fMGL strategy should find broad applications in the tissue imaging of glycans and surgical pathology.
Assuntos
Encéfalo , Química Click , Polissacarídeos , Animais , Camundongos , Humanos , Polissacarídeos/química , Polissacarídeos/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Ácidos Siálicos/metabolismo , Ácidos Siálicos/química , Ácidos Siálicos/análiseRESUMO
Modulation of sialic acids is one of the important pathological consequences of both type 1 and type 2 diabetes mellitus with or without the micro- and macrovascular complications. However, the mechanistic, therapeutic and/or diagnostic implications of these observations are uncoordinated and possibly conflicting. This review critically analyses the scientific investigations connecting sialic acids with diabetes mellitus. Generally, variations in the levels and patterns of sialylation, fucosylation and galactosylation were predominant across various tissues and body systems of diabetic patients, but the immune system seemed to be most affected. These might be explored as a basis for differential diagnosis of various diabetic complications. Sialic acids are predominantly elevated in nearly all forms of diabetic conditions, particularly nephropathy and retinopathy, which suggests some diagnostic value but the mechanistic details were not unequivocal from the available data. The plausible mechanistic explanations for the elevated sialic acids are increased desialylation by sialidases, stimulation of hexosamine pathway and synthesis of acute phase proteins as well as oxidative stress. Additionally, sialic acids are also profoundly associated with glucose transport and insulin resistance in human-based studies while animal-based studies revealed that the increased desialylation of insulin receptors by sialidases, especially NEU1, might be the causal link. Interestingly, inhibition of the diabetes-associated NEU1 desialylation was beneficial in diabetes management and might be considered as a therapeutic target. It is hoped that the article will provide an informed basis for future research activities on the exploitation of sialic acids and glycobiology for therapeutic and/or diagnostic purposes against diabetes mellitus.
Assuntos
Ácidos Siálicos , Humanos , Ácidos Siálicos/metabolismo , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/terapia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/diagnósticoRESUMO
Sialylation during N-glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N-glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N-acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture.
Assuntos
Cricetulus , Fragmentos Fc das Imunoglobulinas , Lactose , Animais , Células CHO , Lactose/análogos & derivados , Lactose/metabolismo , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Cricetinae , Glicosilação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Glicoproteínas/metabolismo , Glicoproteínas/genética , Meios de Cultura/química , Ácidos Siálicos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , OligossacarídeosRESUMO
Polysialylated neural cell adhesion molecule (PSA-NCAM) is expressed in the developing central nervous system (CNS) and plays an important role in neurogenesis. Organophosphorus (OP) toxins, including diazinon (DZN), cause oxidative stress (OS) and damage the CNS. Resveratrol (RV), with its antioxidant effect, leads to the reduction of OS. Therefore, this research was conducted with the aim of the effect of RVon the expression of PSA-NCAM in the hippocampus (HPC) of rat fetuses treated with DZN. In this study, 24 female Wistar rats were divided into 4 groups (n = 6): Control, DZN (40 mg/kg), RV(10 mg/kg), and DZN + RV(40 mg/kg + 10 mg/kg) after confirming they were pregnant. On the 21st day of pregnancy, the mother mice were anesthetized with ketamine and xylazine, and the fetuses were removed; after anesthesia, their brains were removed for immunohistochemistry and western blot (WB) technique. The results of the study showed that in the group receiving DZN, the level of PSA-NCAM protein expression decreased significantly compared to the control group, and the group receiving RV with its antioxidant property increased the expression of PSA-NCAM protein compared to the DZN group. All in all, the exposure of pregnant mice to DZN causes disorders in the CNS, especially the level of PSA-NCAM protein expression in the HPC of fetuses, and the use of RV as an antioxidant by pregnant mothers neutralizes the effects of DZN in the HPC of their fetuses.
Assuntos
Antioxidantes , Diazinon , Hipocampo , Molécula L1 de Adesão de Célula Nervosa , Ratos Wistar , Resveratrol , Ácidos Siálicos , Animais , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Feminino , Diazinon/toxicidade , Gravidez , Resveratrol/farmacologia , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Ácidos Siálicos/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Ratos , Feto/efeitos dos fármacos , Feto/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Inseticidas/toxicidadeRESUMO
Women affected by different autoimmune diseases and displaying positivity for anti-Ro/SSA and anti-La/SSB autoantibodies are at high risk of adverse pregnancies in which placental dysfunction seems to play a determinant role. Sialylation is known to have important implications in the maintenance of the normal morpho-functional features of the placenta. Hence, the present study aimed to investigate possible changes in the distribution and content of sialic acids (Sias) with different glycosidic linkages (i.e., α2,3 and α2,6 Galactose- or N-acetyl-Galactosamine-linked Sias, and polysialic acid) in placentas from anti-Ro/SSA- and anti-La/SSB-positive pregnant women with autoimmune diseases by using lectin histochemistry and polysialic acid immunohistochemistry. Our findings revealed lower levels of α2,3-linked Sias in the trophoblast and basement membrane and/or basal plasma membrane of the pathological cases respect to control placentas. Some vessels of the pathological cases displayed α2,3-linked Sias. α2,6-linked Sias positivity was detected in the trophoblast and in some vessels of the pathological cases, while in control samples it was present only in the vessels. Lower levels of polysialic acid were observed in the trophoblast of pathological cases compared to controls. Collectively, our findings suggest that multiple changes in the sialylation status of placenta might affect placental morpho-functional features in anti-Ro/SSA- and anti-La/SSB-positive pregnancies.
Assuntos
Placenta , Ácidos Siálicos , Humanos , Feminino , Gravidez , Placenta/metabolismo , Ácidos Siálicos/metabolismo , Adulto , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Anticorpos Antinucleares/metabolismo , Trofoblastos/metabolismo , Trofoblastos/patologiaRESUMO
We describe a facile method to prepare water-compatible molecularly imprinted polymer nanogels (MIP NGs) as synthetic antibodies against target glycans. Three different phenylboronic acid (PBA) derivatives were explored as monomers for the synthesis of MIP NGs targeting either α2,6- or α2,3-sialyllactose, taken as oversimplified models of cancer-related sT and sTn antigens. Starting from commercially available 3-acrylamidophenylboronic acid, also its 2-substituted isomer and the 5-acrylamido-2-hydroxymethyl cyclic PBA monoester derivative were initially evaluated by NMR studies. Then, a small library of MIP NGs imprinted with the α2,6-linked template was synthesized and tested by mobility shift Affinity Capillary Electrophoresis (msACE), to rapidly assess an affinity ranking. Finally, the best monomer 2-acrylamido PBA was selected for the synthesis of polymers targeting both sialyllactoses. The resulting MIP NGs display an affinity constant≈106â M-1 and selectivity towards imprinted glycans. This general procedure could be applied to any non-modified carbohydrate template possessing a reducing end.
Assuntos
Ácidos Borônicos , Lactose , Nanogéis , Ácidos Borônicos/química , Lactose/química , Lactose/análogos & derivados , Nanogéis/química , Polímeros Molecularmente Impressos/química , Impressão Molecular , Polímeros/química , Eletroforese Capilar , Polietilenoglicóis/química , Polissacarídeos/química , Ácidos SiálicosRESUMO
We compared the duration of fever in children infected with A(H1N1)pdm09, A(H3N2), or influenza B viruses following treatment with baloxavir marboxil (baloxavir) or neuraminidase inhibitors (NAIs) (oseltamivir, zanamivir, or laninamivir). This observational study was conducted at 10 outpatient clinics across 9 prefectures in Japan during the 2012-2013 and 2019-2020 influenza seasons. Patients with influenza rapid antigen test positive were treated with one of four anti-influenza drugs. The type/subtype of influenza viruses were identified from MDCK or MDCK SIAT1 cell-grown samples using two-step real-time PCR. Daily self-reported body temperature after treatment were used to evaluate the duration of fever by treatment group and various underlying factors. Among 1742 patients <19 years old analyzed, 452 (26.0%) were A(H1N1)pdm09, 827 (48.0%) A(H3N2), and 463 (26.0%) influenza B virus infections. Among fours treatment groups, baloxavir showed a shorter median duration of fever compared to oseltamivir in univariate analysis for A(H1N1)pdm09 virus infections (baloxavir, 22.0 h versus oseltamivir, 26.7 h, P < 0.05; laninamivir, 25.5 h, and zanamivir, 25.0 h). However, this difference was not significant in multivariable analyses. For A(H3N2) virus infections, there were no statistically significant differences observed (20.3, 21.0, 22.0, and 19.0 h) uni- and multivariable analyses. For influenza B, baloxavir shortened the fever duration by approximately 15 h than NAIs (20.3, 35.0, 34.3, and 34.1 h), as supported by uni- and multivariable analyses. Baloxavir seems to have comparable clinical effectiveness with NAIs on influenza A but can be more effective for treating pediatric influenza B virus infections than NAIs.
Assuntos
Antivirais , Dibenzotiepinas , Febre , Guanidinas , Vírus da Influenza A Subtipo H1N1 , Vírus da Influenza A Subtipo H3N2 , Vírus da Influenza B , Influenza Humana , Morfolinas , Oseltamivir , Piranos , Piridonas , Ácidos Siálicos , Triazinas , Zanamivir , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Antivirais/uso terapêutico , Antivirais/farmacologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/genética , Criança , Zanamivir/uso terapêutico , Zanamivir/análogos & derivados , Zanamivir/farmacologia , Triazinas/uso terapêutico , Triazinas/farmacologia , Guanidinas/uso terapêutico , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Piridonas/uso terapêutico , Dibenzotiepinas/uso terapêutico , Japão , Feminino , Masculino , Pré-Escolar , Oseltamivir/uso terapêutico , Febre/tratamento farmacológico , Febre/virologia , Adolescente , Morfolinas/uso terapêutico , Lactente , Estações do Ano , Tiepinas/uso terapêutico , Tiepinas/farmacologia , Oxazinas/uso terapêutico , Fatores de Tempo , Benzoxazinas/uso terapêuticoRESUMO
In individuals with hearing loss, protection of residual hearing is essential following cochlear implantation to facilitate acoustic and electric hearing. Hearing preservation requires slow insertion, atraumatic electrode and delivery of the optimal quantity of a pharmacological agent. Several studies have reported variable hearing outcomes with osmotic pump-mediated steroid delivery. New drugs, such as sialyllactose (SL) which have anti-inflammatory effect in many body parts, can prevent tissue overgrowth. In the present study, the positive effects of the pharmacological agent SL against insults were evaluated in vitro using HEI-OC1 cells. An animal model to simulate the damage due to electrode insertion during cochlear implantation was used. SL was delivered using osmotic pumps to prevent loss of the residual hearing in this animal model. Hearing deterioration, tissue fibrosis and ossification were confirmed in this animal model. Increased gene expressions of inflammatory cytokines were identified in the cochleae following dummy electrode insertion. Following the administration of SL, insertion led to a decrease in hearing threshold shifts, tissue reactions, and inflammatory markers. These results emphasize the possible role of SL in hearing preservation and improve our understanding of the mechanism underlying hearing loss after cochlear implantation.
Assuntos
Implante Coclear , Perda Auditiva , Lactose , Animais , Lactose/análogos & derivados , Lactose/farmacologia , Perda Auditiva/prevenção & controle , Perda Auditiva/tratamento farmacológico , Audição/efeitos dos fármacos , Cóclea/efeitos dos fármacos , Cóclea/metabolismo , Camundongos , Modelos Animais de Doenças , Linhagem Celular , Citocinas/metabolismo , Masculino , Ácidos SiálicosRESUMO
Aberrant sialylation with overexpression of the homopolymeric glycan polysialic acid (polySia) was recently reported in fibroblasts from fibrotic skin lesions. Yet, whether such a rise in polySia levels or sialylation in general may be functionally implicated in profibrotic activation of fibroblasts and their transition to myofibroblasts remains unknown. Therefore, we herein explored whether inhibition of sialylation could interfere with the process of skin fibroblast-to-myofibroblast transition induced by the master profibrotic mediator transforming growth factor ß1 (TGFß1). Adult human skin fibroblasts were pretreated with the competitive pan-sialyltransferase inhibitor 3-Fax-peracetyl-Neu5Ac (3-Fax) before stimulation with recombinant human TGFß1, and then analyzed for polySia expression, cell viability, proliferation, migratory ability, and acquisition of myofibroblast-like morphofunctional features. Skin fibroblast stimulation with TGFß1 resulted in overexpression of polySia, which was effectively blunted by 3-Fax pre-administration. Pretreatment with 3-Fax efficiently lessened TGFß1-induced skin fibroblast proliferation, migration, changes in cell morphology, and phenotypic and functional differentiation into myofibroblasts, as testified by a significant reduction in FAP, ACTA2, COL1A1, COL1A2, and FN1 gene expression, and α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein levels, as well as a reduced contractile capability. Moreover, skin fibroblasts pre-administered with 3-Fax displayed a significant decrease in Smad3-dependent canonical TGFß1 signaling. Collectively, our in vitro findings demonstrate for the first time that aberrant sialylation with increased polySia levels has a functional role in skin fibroblast-to-myofibroblast transition and suggest that competitive sialyltransferase inhibition might offer new therapeutic opportunities against skin fibrosis.