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It is known that bioactivities of chitooligosaccharide (COS) are closely related to the degree of polymerization (DP); therefore, it is essential to prepare COS with controllable DP, such as chitobiose showing high antioxidant and antihyperlipidemia activities. In this study, BLAST, sequence alignment and phylogenetic analysis of characterized glycoside hydrolase (GH) 46 endo-chitosanases revealed that a chitosanase Sn1-CSN from Streptomyces niveus was different from others. Sn1-CSN was overexpressed in E. coli, purified and characterized in detail. It showed the highest activity at pH 6.0 and exhibited superior stability between pH 4.0 and pH 11.0. Sn1-CSN displayed the highest activity at 50 °C and was fairly stable at ≤45 °C. Its apparent kinetic parameters against chitosan (DDA: degree of deacetylation, >94%) were determined, with Km and kcat values of 1.8 mg/mL and 88.3 s-1, respectively. Cu2+ enhanced the activity of Sn1-CSN by 54.2%, whereas Fe3+ inhibited activity by 15.1%. Hydrolysis products of chitosan (DDA > 94%) by Sn1-CSN were mainly composed of chitobiose (87.3%), whereas partially acetylated chitosan with DDA 69% was mainly converted into partially acetylated COS with DP 2-13. This endo-chitosanase has great potential to be used for the preparation of chitobiose and partially acetylated COS with different DPs.
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Glicosídeo Hidrolases , Streptomyces/enzimologia , Sequência de Aminoácidos , Quitosana/química , Quitosana/metabolismo , Dissacarídeos/metabolismo , Escherichia coli/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Metais/química , Simulação de Acoplamento Molecular , Filogenia , TemperaturaRESUMO
It is proven that ß-amyloid (Aß) aggregates containing cross-ß-sheet structures led to oxidative stress, neuroinflammation, and neuronal loss via multiple pathways. Therefore, reduction of Aß neurotoxicity via inhibiting aggregation of Aß or dissociating toxic Aß aggregates into nontoxic forms might be effective therapeutic methods for Alzheimer's disease (AD) treatment. This study was designed to explore interference of chitosan oligosaccharides (COS) on ß-(1-42)-amyloid protein (Aß42) aggregation and Aß42-induced cytotoxicity. Here it was demonstrated that COS showed good blood-brain barrier (BBB) penetration ability in vitro and in vivo. The experimental results showed that COS efficiently interfered with Aß42 aggregation in dose- and degree of polymerization (DP)-dependent manners, and COS monomer with DP6 showed the best effect on preventing conformational transition into ß-sheet-rich structures. Based on the binding affinity analysis by microscale thermophoresis (MST), it was confirmed that COS could directly bind with Aß42 in a DP-dependent manner. Our findings demonstrated that different performance of COS monomers with different DPs against Aß42 assembly was, to some extent, attributable to their different binding capacities with Aß42. As a result, COS significantly ameliorated Aß42-induced cytotoxicity. Taken together, our studies would point towards a potential role of COS in treatment of AD.
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Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Quitosana/química , Oligossacarídeos/administração & dosagem , Fragmentos de Peptídeos/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligossacarídeos/farmacocinética , Oligossacarídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Polimerização , Distribuição TecidualRESUMO
The formation of bacterial biofilms has increased the resistance of bacteria to various environmental factors and is tightly associated with many persistent and chronic bacterial infections. Herein we design a strategy conjugating florfenicol, an antibiotic commonly used in the treatment of streptococcus, with the antimicrobial biomaterial, chitosan oligosaccharides. The results demonstrated that the florfenicol-COS conjugate (F-COS) efficiently eradicated the mature Streptococcus hyovaginalis biofilm, apparently inhibiting drug resistance to florfenicol. A quantity of 250 µg/mL F-COS showed effective inhibitory activity against planktonic cells and biofilm of the bacteria, and a 4-fold improvement of the F-COS compared to unmodified florfenicol was observed. Furthermore, the conjugate showed a broad-spectrum activity against both Gram-positive and Gram-negative bacteria. It suggested that F-COS might have a potential for application in the treatment of biofilm-related infections.
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Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Quitosana/química , Farmacorresistência Bacteriana/efeitos dos fármacos , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Infecções Bacterianas/tratamento farmacológico , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Plâncton/efeitos dos fármacos , Streptococcus/efeitos dos fármacos , Tianfenicol/análogos & derivados , Tianfenicol/química , Tianfenicol/farmacologiaRESUMO
Bacteria biofilm helps bacteria prevent phagocytosis during infection and increase resistance to antibiotics. Staphylococcus aureus is a Gram-positive pathogenic bacterium and is tightly associated with biofilm-related infections, which have led to great threat to human health. Chitosan, the only cationic polysaccharide in nature, has been demonstrated to have antimicrobial and anti-biofilm activities, which, however, require a relative high dosage of chitosan. Moreover, poor water solubility further restricts its applications on anti-infection therapy. Inulins are a group of polysaccharides produced by many types of plants, and are widely used in processed foods. Compared to chitosan, inulin is very soluble in water and possesses a mild antibacterial activity against certain pathogenic bacteria. In order to develop an effective strategy to treat biofilm-related infections, we introduce a method by covalent conjugation of inulin to chitosan. The physicochemical characterization of the inulinâ»chitosan conjugate was assayed, and the anti-biofilm activity was evaluated against S. aureus biofilm. The results indicated that, as compared to chitosan, this novel polysaccharideâ»polysaccharide conjugate significantly enhanced activities against S. aureus either in a biofilm or planktonic state. Of note, the conjugate also showed a broad spectrum anti-biofilm activity on different bacteria strains and low cellular toxicity to mammalian cells. These results suggested that chitosan conjugation of inulin was a viable strategy for treatment against biofilm-related infections. This finding may further spread the application of natural polysaccharides on treatments of infectious disease.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Quitosana/química , Inulina/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Camundongos , Testes de Sensibilidade Microbiana/métodos , Células RAW 264.7 , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacosRESUMO
Chitosan oligosaccharides (COS) display various biological activities. In this study, we aimed to explore the preventive effects of COS on glucolipid metabolism disorder using palmitic acid (PA)-induced HepG2 cells and high-fat diet (HFD)-fed C57BL/6J mice as experimental models in vitro and in vivo, respectively. The results showed that COS pretreatment for 12 h significantly ameliorated lipid accumulation in HepG2 cells exposed to PA for 24 h, accompanied by a reversing of the upregulated mRNA expression of proinflammatory cytokines (IL-6, MCP-1, TNF-α) and glucolipid metabolism-related regulators (SCD-1, ACC1, PCK1-α). In addition, COS treatment alleviated glucolipid metabolism disorder in mice fed with HFD for five months, including reduction in body weight and fasting glucose, restoration of intraperitoneal glucose tolerance, and suppression of overexpression of proinflammatory cytokines and glucolipid metabolism-related regulators. Furthermore, our study found that COS pretreatment significantly reversed the downregulation of PPARγ at transcriptional and translational levels in both PA-induced HepG2 cells and liver tissues of HFD-fed mice. In summary, the study suggests that COS can improve glucolipid metabolism disorder by suppressing inflammation and upregulating PPARγ expression. This indicates a novel application of COS in preventing and treating glucolipid metabolism-related diseases.
Assuntos
Quitosana/farmacologia , Glicolipídeos/metabolismo , Síndrome Metabólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Oligossacarídeos/farmacologia , Animais , Quitosana/química , Quitosana/uso terapêutico , Citocinas/imunologia , Citocinas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Células Hep G2 , Humanos , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Síndrome Metabólica/etiologia , Síndrome Metabólica/imunologia , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/imunologia , Obesidade/metabolismo , Oligossacarídeos/química , Oligossacarídeos/uso terapêutico , PPAR gama/metabolismo , Resultado do Tratamento , Regulação para CimaRESUMO
Gut microbiota has been proved to be an indispensable link between nutrient excess and metabolic syndrome, and chitin oligosaccharide (NACOS) has displayed therapeutic effects on multiple diseases such as cancer and gastritis. In this study, we aim to confirm whether NACOS can ameliorate high-fat diet (HFD)-induced metabolic syndrome by rebuilding the structure of the gut microbiota community. Male C57BL/6J mice fed with HFD were treated with NACOS (1 mg/mL) in drinking water for five months. The results indicate that NACOS improved glucose metabolic disorder in HFD-fed mice and suppressed mRNA expression of the protein regulators related to lipogenesis, gluconeogenesis, adipocyte differentiation, and inflammation in adipose tissues. Additionally, NACOS inhibited the destruction of the gut barrier in HFD-treated mice. Furthermore, 16S ribosome RNA sequencing of fecal samples demonstrates that NACOS promoted the growth of beneficial intestinal bacteria remarkably and decreased the abundance of inflammogenic taxa. In summary, NACOS partly rebuilt the microbial community and improved the metabolic syndrome of HFD-fed mice. These data confirm the preventive effects of NACOS on nutrient excess-related metabolic diseases.
Assuntos
Quitina/administração & dosagem , Dieta Hiperlipídica/efeitos adversos , Microbioma Gastrointestinal/efeitos dos fármacos , Síndrome Metabólica/tratamento farmacológico , Oligossacarídeos/administração & dosagem , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/microbiologia , Lipogênese/efeitos dos fármacos , Masculino , Síndrome Metabólica/microbiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The authors wish to correct Figure 1 in this paper [1] to be as follows:[...].
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Chitosan oligosaccharides (COS), the degraded products of chitosan, have been demonstrated to have versatile biological functions. In primary studies, it has displayed significant adjuvant effects when mixed with other vaccines. In this study, chitosan oligosaccharides with different deacetylation degrees were prepared and conjugated to porcine circovirus type 2 (PCV2) subunit vaccine to enhance its immunogenicity. The vaccine conjugates were designed by the covalent linkage of COSs to PCV2 molecules and administered to BALB/c mice three times at two-week intervals. The results indicate that, as compared to the PCV2 group, COS-PCV2 conjugates remarkably enhanced both humoral and cellular immunity against PCV2 by promoting lymphocyte proliferation and initiating a mixed T-helper 1 (Th1)/T-helper 2 (Th2) response, including raised levels of PCV2-specific antibodies and an increased production of inflammatory cytokines. Noticeably, with the increasing deacetylation degree, the stronger immune responses to PCV2 were observed in the groups with COS-PCV2 vaccination. In comparison with NACOS (chitin oligosaccharides)-PCV2 and LCOS (chitosan oligosaccharides with low deacetylation degree)-PCV2, HCOS (chitosan oligosaccharides with high deacetylation degree)-PCV2 showed the highest adjuvant effect, even comparable to that of PCV2/ISA206 (a commercialized adjuvant) group. In summary, COS conjugation might be a viable strategy to enhance the immune response to PCV2 subunit vaccine, and the adjuvant effect was positively correlated with the deacetylation degree of COS.
Assuntos
Quitosana/imunologia , Circovirus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Imunidade Celular/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Conjugadas/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Virais/farmacologiaRESUMO
Antrodia cinnamomea (AC), an edible fungus growing in Taiwan, has various health benefits. This study was designed to examine the potential inhibitory effects of AC oligosaccharides on lipopolysaccharide (LPS)-induced inflammatory responses in vitro and in vivo. By trifluoroacetic acid degradation, two oligosaccharide products were prepared from AC polysaccharides at 90 °C (ACHO) or 25 °C (ACCO), which showed different oligosaccharide identities. Compared to ACCO, ACHO displayed better inhibitory effects on LPS-induced mRNA expression of pro-inflammatory cytokines including IL-6, IL-8, IL-1ß, TNF-α and MCP-1 in macrophage cells. Further, ACHO significantly suppressed the inflammation in lung tissues of LPS-injected C57BL/6 mice. The potential anti-inflammatory molecular mechanism may be associated with the promotion of protein O-GlcNAcylation, which further skewed toward the marked suppression of p38 and Akt phosphorylation. Our results suggest that the suppressive effect of AC oligosaccharides on inflammation may be an effective approach for the prevention of inflammation-related diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Inflamação/tratamento farmacológico , Lipopolissacarídeos/metabolismo , Oligossacarídeos/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antrodia , Citocinas/metabolismo , Descoberta de Drogas/métodos , Humanos , Inflamação/induzido quimicamente , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , RNA Mensageiro , Transdução de Sinais , Fator de Necrose Tumoral alfaRESUMO
Eukaryotic sialyltransferases play key roles in many physiological and pathological events. The expression of active human recombinant sialyltransferases in bacteria is still challenging. In the current study, the genes encoding human N-acetylgalactosaminide α2,6-sialyltransferase V (hST6GalNAc V) and N-acetylgalactosaminide α2,6-sialyltransferase VI (hST6GalNAc VI) lacking the N-terminal transmembrane domains were cloned into the expression vectors, pET-32a and pET-22b, respectively. Soluble and active forms of recombinant hST6GalNAc V and hST6GalNAc VI when coexpressed with the chaperone plasmid pGro7 were successfully achieved in Escherichia coli. Further, lactose (Lac), Lacto-N-triose II (LNT II), lacto-N-tetraose (LNT), and sialyllacto-N-tetraose a (LSTa) were used as acceptor substrates to investigate their activities and substrate specificities. Unexpectedly, both can transfer sialic acid onto all those substrates. Compared with hST6GalNAc V expressed in the mammalian cells, the recombinant two α2,6-sialyltransferases in bacteria displayed flexible substrate specificities and lower enzymatic efficiency. In addition, an important human milk oligosaccharide disialyllacto-N-tetraose (DSLNT) can be synthesized by both human α2,6-sialyltransferases expressed in E. coli using LSTa as an acceptor substrate. To the best of our knowledge, these two active human α2,6-sialyltransferases enzymes were expressed in bacteria for the first time. They showed a high potential to be applied in biotechnology and investigating the molecular mechanisms of biological and pathological interactions related to sialylated glycoconjugates.
Assuntos
Escherichia coli , Proteínas Recombinantes , Sialiltransferases , Humanos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Lactose/metabolismo , Oligossacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Sialiltransferases/genética , Sialiltransferases/metabolismo , Especificidade por SubstratoRESUMO
Chitosan and chitooligosaccharide (COS) are renowned for their potent antimicrobial prowess, yet the precise antimicrobial efficacy of COS remains elusive due to scant structural information about the utilized saccharides. This study delves into the antimicrobial potential of COS, spotlighting a distinct hetero-chitooligosaccharide dubbed DACOS. In contrast to other COS, DACOS remarkably fosters the growth of Candida tropicalis planktonic cells and fungal biofilms. Employing gradient alcohol precipitation, DACOS was fractionated, unveiling diverse structural characteristics and differential impacts on C. tropicalis. Notably, in a murine model of systemic candidiasis, DACOS, particularly its 70 % alcohol precipitates, manifests a promotive effect on Candida infection. This research unveils a new pathway for exploring the intricate nexus between the structural attributes of chitosan oligosaccharides and their physiological repercussions, underscoring the imperative of crafting chitosan and COS with meticulously defined structural configurations.
Assuntos
Anti-Infecciosos , Quitosana , Oligossacarídeos , Animais , Camundongos , Candida tropicalis , Quitosana/farmacologia , Quitosana/química , Antifúngicos/farmacologia , BiofilmesRESUMO
The biofilms formed by pathogenic microorganisms seriously threaten human health and significantly enhance drug resistance, which urgently call for developing drugs specifically targeting on biofilms. Chitooligosaccharides extracted from shrimp and crab shells are natural alkaline oligosaccharides with excellent antibacterial effects. Nevertheless, their inhibition efficacy on biofilms still needs to be improved. Spirulina (SP) is a microalga with negatively charged surface, and its spiral structure facilitates colonization in the depth of the biofilm. Therefore, the complex of Spirulina and chitooligosaccharides may play a synergistic role in killing pathogens in the depth of biofilm. This research first screened chitooligosaccharides with significant bactericidal effects. Subsequently, Spirulina@Chitooligosaccharides (SP@COS complex was prepared by combining chitooligosaccharides with Spirulina through electrostatic adsorption. The binding of the complex was characterized by zeta potential, z-average size, and fluorescence labeling. Ultraviolet-visible spectroscopy (UV-Vis) showed the encapsulation efficiency and the drug loading efficiency reached up to 90% and 16%, respectively. The prepared SP@COS2 exhibited a profound synergistic inhibition effect on bacterial and fungal biofilms, which was mainly achieved by destroying the cell structure of the biofilm. These results demonstrate the potential of Spirulina-chitooligosaccharides complex as a biofilm inhibitor and provide a new idea for addressing the harm of pathogenic microorganisms.
Assuntos
Quitosana , Spirulina , Humanos , Antibacterianos/farmacologia , Antibacterianos/química , Quitosana/farmacologia , Biofilmes , Quitina/farmacologiaRESUMO
Glycocalyx dysfunction is believed as the first step in diabetic vascular disease. However, few studies have systematically investigated the influence of HG on the glycocalyx as a whole and its major constituent glycans towards one type of cell. Furthermore, most studies utilized traditional two-dimensional (2D) cultures in vitro, which can't provide the necessary fluid environment for glycocalyx. Here, we utilized vascular glycocalyx on chips to evaluate the changes of glycocalyx and its constituent glycans in HG induced HUVECs. Fluorescence microscopy showed up-regulation of hyaluronan (HA) but down-regulation of heparan sulfate (HS). By analyzing the metabolic enzymes of both glycans, a decrease in the ratio of synthetic/degradative enzymes for HA and an increase in that for HS were demonstrated. Two substrates (UDP-GlcNAc, UDP-GlcA) for the synthesis of both glycans were increased according to omics analysis. Since they were firstly pumped into Golgi apparatus to synthesize HS, less substrates may be left for HA synthesis. Furthermore, the differential changes of HA and HS were confirmed in vessel slides from db/db mice. This study would deepen our understanding of impact of HG on glycocalyx formation and diabetic vascular disease.
Assuntos
Angiopatias Diabéticas , Ácido Hialurônico , Camundongos , Animais , Ácido Hialurônico/metabolismo , Heparitina Sulfato/metabolismo , Glucose , Difosfato de UridinaRESUMO
Osteoarthritis (OA) is a degenerative joint disease, which can lead to joint pain, stiffness, deformity and dysfunction, that seriously affects the quality of life in patients. At present, the treatments of OA mainly include early pharmacological treatment and late joint replacement. However, current pharmacological treatment has limited efficacy and undesired side effects. Chitosan oligosaccharide (COS) is a kind of nontoxic and biodegradable oligo-saccharide, which is composed of 2-20 glucosamine or N-acetylglucosamine linked by ß-1,4 glycosidic bond. Studies have shown that COS has significant biological properties like antimicrobial, anti-inflammatory, antioxidant, and anti-tumor, as well as immunoregulation ability. However, the effects of COS on OA have not been clarified. In this study, we explored the protective effects of COS with different degrees of deacetylation on chondrocytes stimulated by interleukin 1ß (IL-1ß) in vitro. The results showed that IL-1ß inhibited cell proliferation and promoted cell apoptosis. Besides that, IL-1ß increased the expression of the major chondro-degrading genes MMP13 and ADAMTS-5, while decreased the expression of COL2A and ACAN. COS with different degrees of deacetylation (HDACOS, MDACOS, LDACOS) had different effects on IL-1ß induced inflammation. LDACOS had the most obvious anti-inflammatory effects to inhibit the expression of MMP13 and ADAMTS-5 while promoted the expression of COL2A and ACAN. In addition, we found that the expression of autophagy-related gene Beclin-1 was up-regulated, and the ratio of LC3-II/LC3-I was increased in the LDACOS group. Furthermore, transmission electron microscopy (TEM) analysis showed that the number of intracellular autophagosomes increased significantly with the treatment of LDACOS. Based on our research, we suggested that LDACOS could inhibit chondrocytes inflammation and promote cell autophagy, and might be a protective drug for the treatment of OA.
Assuntos
Quitosana , Osteoartrite , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Autofagia , Quitosana/metabolismo , Quitosana/farmacologia , Condrócitos , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Interleucina-1beta/uso terapêutico , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Oligossacarídeos/farmacologia , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologia , Qualidade de VidaRESUMO
Past studies on the protective effects of chitosan oligosaccharides (COS) on inflammatory bowel disease (IBD) commonly rely on animal models, because traditional cell culture systems couldn't faithfully mimic human intestinal physiology. Here a novel human gut-on-a-chip microsystem was established to further explore the regulatory effects of COS on the occurrence and development of human enteritis. By constructing an intestinal injury model caused by dextran sodium sulfate (DSS) on the chip, this study proved that COS can reduce intestinal epithelial injury by promoting the expression of the mucous layer for the first time. By establishing an inflammatory bowel disease model on the chip caused by E. coli 11775, this study demonstrated that COS can protect the intestinal epithelial barrier and vascular endothelial barrier by inhibiting the adhesion and invasion of E. coli 11775 for the first time. In addition, similar to the results in vivo, COS can decrease the inflammatory response by reducing the expression of toll-like receptor 4 protein and reducing the nuclear DNA binding rate of nuclear factor kappa-B protein on this chip. In summary, COS can be used as a potential drug to treat human IBD and the human gut-on-a-chip would be used as a platform for quick screening drugs to treat human IBD in future.
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The pandemic caused by SARS-CoV-2 is the most widely spread disease in the 21st century. Due to the continuous emergence of variants across the world, it is necessary to expand our understanding of host-virus interactions and explore new agents against SARS-CoV-2. In this study, it was found exopolysaccharides (EPSs) from halophilic archaeon Haloarcula hispanica ATCC33960 can bind to the spike protein of SARS-CoV-2 with the binding constant KD of 2.23 nM, block the binding of spike protein to Vero E6 and bronchial epithelial BEAS-2B cells, and inhibit pseudovirus infection. However, EPSs from the gene deletion mutant â³HAH_1206 almost completely lost the antiviral activity against SARS-CoV-2. A significant reduction of glucuronic acid (GlcA) and the sulfation level in EPSs of â³HAH_1206 was clearly observed. Our results indicated that sulfated GlcA in EPSs is possible for a main structural unit in their inhibition of binding of SARS-CoV-2 to host cells, which would provide a novel antiviral mechanism and a guide for designing new agents against SARS-CoV-2.
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The fungal cell wall is an ideal target for the design of antifungal drugs. In this study we used an analog of cell wall polymer, a highly deacetylated high molecular-weight chitosan oligosaccharide (HCOS), to test its effect against pathogenic Candida strains. Results showed that HCOS was successfully incorporated into the dynamic cell wall organization process and exhibited an apparent antifungal activity against both plankton and mature fungal biofilm, by impairing the cell wall integrity. Unexpectedly, mechanistic studies suggested that HCOS exerts its activity by interfering with family members of PHR ß-(1,3)-glucanosyl transferases and affecting the connection and assembly of cell wall polysaccharides. Furthermore, HCOS showed great synergistic activity with different fungicides against Candida cells, especially those in biofilm. These findings indicated HCOS has a great potential as an antifungal drug or drug synergist and proposed a novel antifungal strategy with structure-specific oligosaccharides mimicking cell wall polysaccharide fragments.
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Antifúngicos , Quitosana , Antifúngicos/farmacologia , Biofilmes , Candida albicans , Parede Celular , Quitosana/farmacologia , Testes de Sensibilidade Microbiana , Peso Molecular , Oligossacarídeos/farmacologiaRESUMO
Glucosamine (GlcN) is used as a supplement for arthritis and joint pain and has been proved to have effects on inflammation, cancer, and cardiovascular diseases. However, there are limited studies on the regulatory mechanism of GlcN against glucose and lipid metabolism disorder. In this study, we treated high-fat diet (HFD)-induced diabetic mice with GlcN (1 mg/ml, in drinking water) for five months. The results show that GlcN significantly reduced the fasting blood glucose of HFD-fed mice and improved glucose tolerance. The feces of intestinal contents in mice were analyzed using 16s rDNA sequencing. It was indicated that GlcN reversed the imbalanced gut microbiota in HFD-fed mice. Based on the PICRUSt assay, the signaling pathways of glucolipid metabolism and biosynthesis were changed in mice with HFD feeding. By quantitative real-time PCR (qPCR) and hematoxylin and eosin (H&E) staining, it was demonstrated that GlcN not only inhibited the inflammatory responses of colon and white adipose tissues, but also improved the intestinal barrier damage of HFD-fed mice. Finally, the correlation analysis suggests the most significantly changed intestinal bacteria were positively or negatively related to the occurrence of inflammation in the colon and fat tissues of HFD-fed mice. In summary, our studies provide a theoretical basis for the potential application of GlcN to glucolipid metabolism disorder through the regulation of gut microbiota.
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Endo-fucoidanases are important in structural analysis of fucoidans and preparation of fuco-oligosaccharides. However their enzymological properties and analysis of degradation products are scarcely investigated. Truncated endo-α (1 â 3)-fucoidanase Fda1 (tFda1B from Alteromonas sp. was overexpressed and characterized, showing highest activity at pH 7.0, 35 °C, and 1.0 M NaCl. Its Km and kcat were 3.88 ± 0.81 mg/mL and 0.82 ± 0.17 min-1. Fe3+ and Mn2+ enhanced activity by 100% and 19.5% respectively. Co2+ and Cu2+ completely inactivated tFda1B, whereas Ni2+, Mg2+, Zn2+, Pb2+, Ca2+, Ba2+ and Li+ decreased activity by 58.8%, 56.0%, 50.6%, 47.7%, 28.9%, 15.6% and 37.5%, respectively. Catalytic residues were identified through structure and sequence alignment, and confirmed by mutagenesis. Degradation products of Kjellmaniella crassifolia fucoidan by tFda1B were characterized by LC-ESI-MS/MS, confirming tFda1B belongs to endo-(1 â 3)-fucoidanases, and backbone of K. crassifolia fucoidan is 1 â 3 fucoside linkage. This endo-α (1 â 3)-fucoidanase would be useful for elucidating fucoidan structures, and be used as a food enzyme.
Assuntos
Alteromonas/enzimologia , Hidrolases/química , Hidrolases/metabolismo , Polissacarídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Estabilidade Enzimática , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrolases/genética , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Phaeophyceae/química , Phaeophyceae/metabolismo , Filogenia , Polissacarídeos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Organ-on-a-chip as a new technology distinguishes it from animal and cell models at least in three aspects: (1) it responds to drugs' efficacy or toxicity more really by mimicking the human body's fluid microenvironment; (2) it can be used for high throughput screening a large number of compounds; (3) it has physiological accuracy. It is well known that ginsenosides compound K (CK) as a carbohydrate drug has numerous biological activities and physiological functions. However, pharmacokinetic studies of carbohydrate-based CK haven't been performed on organ chips. Here, we established and evaluated the function of single-organ chips and multi-organ chips based on intestinal, vascular, liver, and kidney chips. Each single-organ-on-a-chip performed itself well. Based on organ-on-chips, absorption, metabolism and toxicity of CK were successfully investigated. The pharmacokinetic results of CK provided by chip were consistent with previous reports, demonstrating the reliability of the organ-on-a-chip platform and its potential for use in pharmacokinetic studies of carbohydrate-drugs. As far as we know, this study would be the first report on the pharmacological investigation of carbohydrate drugs on organ-on-a-chip, which provides a theoretical basis for carbohydrate-based drug discovery.