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Multivalent lectin-glycan interactions (MLGIs) are pivotal for viral infections and immune regulation. Their structural and biophysical data are thus highly valuable, not only for understanding their basic mechanisms but also for designing potent glycoconjugate therapeutics against target MLGIs. However, such information for some important MGLIs remains poorly understood, greatly limiting research progress. We have recently developed densely glycosylated nanoparticles, e.g., â¼4 nm quantum dots (QDs) or â¼5 nm gold nanoparticles (GNPs), as mechanistic probes for MLGIs. Using two important model lectin viral receptors, DC-SIGN and DC-SIGNR, we have shown that these probes can not only offer sensitive fluorescence assays for quantifying MLGI affinities, but also reveal key structural information (e.g., binding site orientation and binding mode) useful for MLGI targeting. However, the small sizes of the previous scaffolds may not be optimal for maximising MLGI affinity and targeting specificity. Herein, using α-manno-α-1,2-biose (DiMan) functionalised GNP (GNP-DiMan) probes, we have systematically studied how GNP scaffold size (e.g., 5, 13, and 27 nm) and glycan density (e.g., 100, 75, 50 and 25%) determine their MLGI affinities, thermodynamics, and antiviral properties. We have developed a new GNP fluorescence quenching assay format to minimise the possible interference of GNP's strong inner filter effect in MLGI affinity quantification, revealing that increasing the GNP size is highly beneficial for enhancing MLGI affinity. We have further determined the MLGI thermodynamics by combining temperature-dependent affinity and Van't Hoff analyses, revealing that GNP-DiMan-DC-SIGN/R binding is enthalpy driven with favourable binding Gibbs free energy changes (ΔG°) being enhanced with increasing GNP size. Finally, we show that increasing the GNP size significantly enhances their antiviral potency. Notably, the DiMan coated 27 nm GNP potently and robustly blocks both DC-SIGN and DC-SIGNR mediated pseudo-Ebola virus cellular entry with an EC50 of â¼23 and â¼49 pM, respectively, making it the most potent glycoconjugate inhibitor against DC-SIGN/R-mediated Ebola cellular infections. Our results have established GNP-glycans as a new tool for quantifying MLGI biophysical parameters and revealed that increasing the GNP scaffold size significantly enhances their MLGI affinities and antiviral potencies.
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Antivirais , Ouro , Nanopartículas Metálicas , Polissacarídeos , Termodinâmica , Ouro/química , Nanopartículas Metálicas/química , Humanos , Antivirais/química , Antivirais/farmacologia , Polissacarídeos/química , Lectinas Tipo C/metabolismo , Lectinas Tipo C/química , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/química , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/química , Lectinas/química , Lectinas/metabolismoRESUMO
The dendritic cell tetrameric lectin, DC-SIGN, and its closely related endothelial cell lectin, DC-SIGNR (collectively abbreviated as DC-SIGN/R) play a key role in the binding and transmission of deadly viruses, including Ebola, HIV, HCV, and SARS-CoV-2. Their virus binding/release processes involve a gradually acidifying environment following the natural intracellular trafficking pathways. Therefore, understanding DC-SIGN/R's pH-dependent binding properties with glycan ligands is of great importance. We have recently developed densely glycosylated gold nanoparticles (glycan-GNPs) as a powerful new tool for probing DC-SIGN/R multivalent lectin-glycan interaction (MLGI) mechanisms. They can provide not only quantitative MLGI affinities but also important structural information, such as binding site orientation and binding modes. Herein, we further employ the glycan-GNP probes to investigate the pH dependency of DC-SIGN/R MLGI properties. We find that DC-SIGN/R MLGIs exhibit distinct pH dependence over the normal physiological (7.4) to lysosomal (â¼4.6) pH range. DC-SIGN binds glycan-GNPs strongly and stably from pH 7.4 to â¼5.8, but the binding is weakened significantly as pH decreases to ≤5.4 and may be fully dissociated at pH 4.6. This behaviour is fully consistent with DC-SIGN's role as an endocytic recycling receptor. In contrast, DC-SIGNR's affinity with glycan-GNPs is enhanced with the decreasing pH from 7.4 to 5.4, peaking at pH 5.4, and then reduced as pH is further lowered. Interestingly, both DC-SIGN/R binding with glycan-GNPs are found to be partially reversible in a pH-dependent manner.
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Aspirin, also named acetylsalicylate, can directly acetylate the side-chain of lysine in protein, which leads to the possibility of unexplained drug effects. Here, the study used isotopic-labeling aspirin-d3 with mass spectrometry analysis to discover that aspirin directly acetylates 10 HDACs proteins, including SIRT1, the most studied NAD+-dependent deacetylase. SIRT1 is also acetylated by aspirin in vitro. It is also identified that aspirin directly acetylates lysine 408 of SIRT1, which abolishes SIRT1 deacetylation activity by impairing the substrates binding affinity. Interestingly, the lysine 408 of SIRT1 can be acetylated by CBP acetyltransferase in cells without aspirin supplement. Aspirin can inhibit SIRT1 to increase the levels of acetylated p53 and promote p53-dependent apoptosis. Moreover, the knock-in mice of the acetylation-mimic mutant of SIRT1 show the decreased production of pro-inflammatory cytokines and maintain intestinal immune homeostasis. The study indicates the importance of the acetylated internal functional site of SIRT1 in maintaining intestinal immune homeostasis.
Assuntos
Aspirina , Homeostase , Sirtuína 1 , Sirtuína 1/metabolismo , Sirtuína 1/genética , Animais , Aspirina/farmacologia , Acetilação/efeitos dos fármacos , Camundongos , Homeostase/efeitos dos fármacos , Humanos , Intestinos/efeitos dos fármacos , Camundongos Endogâmicos C57BLRESUMO
Rationale: Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by excessive immune response usually due to lung inflammation. Local immunosuppression is crucial for effective ARDS treatment. However, current methods are limited in their ability to target the lungs specifically. Methods: This study utilized lung-targeted lipid nanoparticles (LNPs) with 1,2-dioleoyl-3-trimethylammonium-propane (termed DOTAP-LNPs) to encapsulate chemically modified soluble programmed death ligand-1 (sPD-L1) mRNA and examined its physiological characteristics and therapeutic efficacy. A comparative analysis was performed between sPD-L1 mRNA delivered by DOTAP-LNPs, sPD-L1 mRNA delivered by regular LNPs (MC3-LNPs), and PD-L1-Fc recombinant protein administered systemically. Additionally, the survival rate of ARDS mice treated with different drugs was assessed. Results: Administration of sPD-L1 mRNA-LNPs to ARDS model mice significantly reduced leukocyte chemotaxis and protein accumulation in lung tissue, along with a decrease in pulmonary edema. Notably, in situ intervention using sPD-L1 mRNA-DOTAP-LNPs exhibited superior therapeutic effects compared to PD-L1-Fc recombinant protein and sPD-L1 mRNA encapsulated in MC3-LNPs. Importantly, treatment with sPD-L1 mRNA-DOTAP-LNPs improved the survival rate of ARDS model mice. Conclusion: This study demonstrates the feasibility of utilizing stable and reliable mRNA to express the immunosuppressive molecule sPD-L1 specifically in the lungs. The findings provide proof of concept for localized nanoparticle delivery and offer a novel therapeutic strategy for treating acute inflammation in ARDS.
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Nanopartículas , Síndrome do Desconforto Respiratório , Animais , Camundongos , Antígeno B7-H1 , Pulmão , Proteínas Recombinantes , Síndrome do Desconforto Respiratório/tratamento farmacológicoRESUMO
Bacterial keratitis is a serious ocular disease that affects millions of people worldwide each year, among which ≈25% are caused by Staphylococcus aureus. With the spread of bacterial resistance, refractory keratitis caused by methicillin-resistant S. aureus (MRSA) affects ≈120 000-190 000 people annually and is a significant cause of infectious blindness. Atomically precise gold nanoclusters (GNCs) recently emerged as promising antibacterial agents; although how the GNC structure and capping ligands control the antibacterial properties remains largely unexplored. In this study, by adjusting the ratio of a "bulky" thiol fragrance to a linear zwitterionic ligand, the GNC conformation is transformed from Au25 (SR)18 to Au23 (SR)16 species, simultaneously converting both inactive thiol ligands into potent antibacterial nanomaterials. Surprisingly, mixed-ligand capped Au23 (SR)16 GNCs exhibit superior antibacterial potency compared to their monoligand counterparts. The optimal GNC is highly potent against MRSA, showing >1024-fold lower minimum inhibitory concentration than the corresponding free ligands. Moreover, it displays excellent potency in treating MRSA-induced keratitis in mice with greatly accelerated corneal recovery (by approximately ninefold). Thus, this study establishes a feasible method to synthesize antibacterial GNCs by adjusting the ligand ratio to control GNC conformation and active non-antibacterial ligands, thereby greatly increasing the repertoires for combating multidrug-resistant bacterial infections.
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Mutations in spike (S) protein epitopes allow SARS-CoV-2 variants to evade antibody responses induced by infection and/or vaccination. In contrast, mutations in glycosylation sites across SARS-CoV-2 variants are very rare, making glycans a potential robust target for developing antivirals. However, this target has not been adequately exploited for SARS-CoV-2, mostly due to intrinsically weak monovalent protein-glycan interactions. We hypothesize that polyvalent nano-lectins with flexibly linked carbohydrate recognition domains (CRDs) can adjust their relative positions and bind multivalently to S protein glycans, potentially exerting potent antiviral activity. Herein, we displayed the CRDs of DC-SIGN, a dendritic cell lectin known to bind to diverse viruses, polyvalently onto 13 nm gold nanoparticles (named G13-CRD). G13-CRD bound strongly and specifically to target glycan-coated quantum dots with sub-nM Kd. Moreover, G13-CRD neutralized particles pseudotyped with the S proteins of Wuhan Hu-1, B.1, Delta variant and Omicron subvariant BA.1 with low nM EC50. In contrast, natural tetrameric DC-SIGN and its G13 conjugate were ineffective. Further, G13-CRD potently inhibited authentic SARS-CoV-2 B.1 and BA.1, with <10 pM and <10 nM EC50, respectively. These results identify G13-CRD as the 1st polyvalent nano-lectin with broad activity against SARS-CoV-2 variants that merits further exploration as a novel approach to antiviral therapy.
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Multivalent lectin-glycan interactions (MLGIs) are widespread in biology and hold the key to many therapeutic applications. However, the underlying structural and biophysical mechanisms for many MLGIs remain poorly understood, limiting our ability to design glycoconjugates to potently target specific MLGIs for therapeutic intervention. Glycosylated nanoparticles have emerged as a powerful biophysical probe for MLGIs, although how nanoparticle shape affects the MLGI molecular mechanisms remains largely unexplored. Herein, we have prepared fluorescent quantum nanorods (QRs), densely coated with α-1,2-manno-biose ligands (QR-DiMan), as multifunctional probes to investigate how scaffold geometry affects the MLGIs of a pair of closely related, tetrameric viral receptors, DC-SIGN and DC-SIGNR. We have previously shown that a DiMan-capped spherical quantum dot (QD-DiMan) gives weak cross-linking interactions with DC-SIGNR but strong simultaneous binding with DC-SIGN. Against the elongated QR-DiMan, DC-SIGN retains similarly strong simultaneous binding of all four binding sites with a single QR-DiMan (apparent K d ≈ 0.5 nM, â¼1.8 million-fold stronger than the corresponding monovalent binding), while DC-SIGNR gives both weak cross-linking and strong individual binding interactions, resulting in a larger binding affinity enhancement than that with QD-DiMan. S/TEM analysis of QR-DiMan-lectin assemblies reveals that DC-SIGNR's different binding modes arise from the different nanosurface curvatures of the QR scaffold. The glycan display at the spherical ends presents too high a steric barrier for DC-SIGNR to bind with all four binding sites; thus, it cross-links between two QR-DiMan to maximize binding multivalency, whereas the more planar character of the cylindrical center allows the glycans to bridge all binding sites in DC-SIGNR. This work thus establishes glycosylated QRs as a powerful biophysical probe for MLGIs not only to provide quantitative binding affinities and binding modes but also to demonstrate the specificity of multivalent lectins in discriminating different glycan displays in solution, dictated by the scaffold curvature.
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Multivalent lectin-glycan interactions (MLGIs) are widespread and vital for biology. Their binding biophysical and structural details are thus highly valuable, not only for the understanding of binding affinity and specificity mechanisms but also for guiding the design of multivalent therapeutics against specific MLGIs. However, effective techniques that can reveal all such details remain unavailable. We have recently developed polyvalent glycan quantum dots (glycan-QDs) as a new probe for MLGIs. Using a pair of closely related tetrameric viral-binding lectins, DC-SIGN and DC-SIGNR, as model examples, we have revealed and quantified their large affinity differences in glycan-QD binding are due to distinct binding modes: with simultaneous binding for DC-SIGN and cross-linking for DC-SIGNR. Herein, we further extend the capacity of the glycan-QD probes by investigating the correlation between binding mode and binding thermodynamics and kinetics and further probing a structural basis of their binding nature. We reveal that while both lectins' binding with glycan-QDs is enthalpy driven with similar binding enthalpy changes, DC-SIGN pays a lower binding entropy penalty, resulting in a higher affinity than DC-SIGNR. We then show that DC-SIGN binding gives a single second-order kon rate, whereas DC-SIGNR gives a rapid initial binding followed by a much slower secondary interaction. We further identify a structural element in DC-SIGN, absent in DC-SIGNR, that plays an important role in maintaining DC-SIGN's MLGI character. Its removal switches the binding from being enthalpically to entropically driven and gives mixed binding modes containing both simultaneous and cross-linking binding behavior, without markedly affecting the overall binding affinity and kinetics.
Assuntos
Pontos Quânticos , Pontos Quânticos/química , Polissacarídeos/química , Cinética , TermodinâmicaRESUMO
Developing effective lymph-node (LN) targeting and imaging probes is crucial for the early detection and diagnosis of tumor metastasis to improve patient survival. Most current clinical LN imaging probes are based on small organic dyes (e.g., indocyanine green) or radioactive 99mTc-complexes, which often suffer from limitations, such as rapid photobleaching, poor signal contrast, and potential biosafety issues. Moreover, these probes cannot easily incorporate therapeutic functions to realize beneficial theranostics without affecting their LN-targeting ability. Herein, we have developed dual-ligand-/multiligand-capped gold nanoclusters (GNCs) for specific targeting, near-infrared (NIR) fluorescence imaging, diagnosis, and treatment of LN cancer metastasis in in vivo mouse models. By optimizing the surface ligand coating, we have prepared Au25(SR1)n(SR2)18-n (where SR1 and SR2 are different functional thiol ligands)-type GNCs, which display highly effective LN targeting, excellent stability and biocompatibility, and optimal body-retention time. Moreover, they can provide continuous NIR fluorescence imaging of LNs for >3 h from a single dose, making them well-suited for fluorescence-guided surgery. Importantly, we have further incorporated methotrexate, a chemotherapeutic drug, into the GNCs without affecting their LN-targeting ability. Consequently, they can significantly improve the efficiency of methotrexate delivery to target LNs, achieving excellent therapeutic efficacy with up to 4-fold lower hepatotoxicity. Thus, the GNCs are highly effective and safe theranostic nanomedicines against cancer lymphatic metastasis.
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Ouro , Verde de Indocianina , Animais , Camundongos , Metástase Linfática/diagnóstico por imagem , Ligantes , Metotrexato , Imagem Óptica/métodos , Corantes , Compostos de SulfidrilaRESUMO
The human mitochondrial alpha-ketoglutarate (α-KG) dehydrogenase complex (hKGDHc) is a well-studied macromolecular enzyme that converts α-KG to succinyl-CoA and NADH. Abnormalities of the complex lead to several diseases, including neurodegenerative disorders. Despite its importance in human metabolism and diseases, structural information on hKGDHc is not well defined. Here, we report the 2.92 Å resolution cryo-electron microscopy (EM) structure of its E1 component 2-oxoglutarate dehydrogenase (OGDH). The density map comprised residues 129-1,023, which is nearly the full length of OGDH. The structure clearly shows the active site and Ca2+ binding site of OGDH. This structural information will improve our understanding of the structure and function of hKGDHc and benefit pharmaceutical and basic science targeting this enzyme complex.
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Complexo Cetoglutarato Desidrogenase , Ácidos Cetoglutáricos , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismoRESUMO
Infections caused by multidrug-resistant (MDR) bacteria are an increasing global healthcare concern. In this study, we developed a dual-ligand-functionalised Au25(SR1) x (SR2)18-x -type gold nanocluster and determined its antibacterial activity against MDR bacterial strains. The pyridinium ligand (SR1) provided bactericidal potency and the zwitterionic ligand (SR2) enhanced the stability and biocompatibility. By optimising the ligand ratio, our gold nanocluster could effectively kill MDR Gram-positive bacteria via multiple antibacterial actions, including inducing bacterial aggregation, disrupting bacterial membrane integrity and potential, and generating reactive oxygen species. Moreover, combining the optimised gold nanocluster with common antibiotics could significantly enhance the antibacterial activity against MDR bacteria both in in vitro and animal models of skin infections. Furthermore, the fluorescence of the gold nanocluster at the second near-infrared (NIR-II) biological window allowed for the monitoring of its biodistribution and body clearance, which confirmed that the gold nanoclusters had good renal clearance and biocompatibility. This study provides a new strategy to combat the MDR challenge using multifunctional gold nanomaterials.
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Charcot-Marie-Tooth disease is the most common inherited peripheral neuropathy. Dominant mutations in the glycyl-tRNA synthetase (GARS) gene cause peripheral nerve degeneration and lead to CMT disease type 2D. The underlying mechanisms of mutations in GARS (GARSCMT2D ) in disease pathogenesis are not fully understood. In this study, we report that wild-type GARS binds the NAD+ -dependent deacetylase SIRT2 and inhibits its deacetylation activity, resulting in the acetylated α-tubulin, the major substrate of SIRT2. The catalytic domain of GARS tightly interacts with SIRT2, which is the most CMT2D mutation localization. However, CMT2D mutations in GARS cannot inhibit SIRT2 deacetylation, which leads to a decrease of acetylated α-tubulin. Genetic reduction of SIRT2 in the Drosophila model rescues the GARS-induced axonal CMT neuropathy and extends the life span. Our findings demonstrate the pathogenic role of SIRT2-dependent α-tubulin deacetylation in mutant GARS-induced neuropathies and provide new perspectives for targeting SIRT2 as a potential therapy against hereditary axonopathies.
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Doença de Charcot-Marie-Tooth/metabolismo , Sirtuína 2/metabolismo , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Drosophila , Glicina-tRNA Ligase/genética , Glicina-tRNA Ligase/metabolismo , Células HEK293 , Humanos , Sirtuína 2/genéticaRESUMO
Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.
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Carboidratos/uso terapêutico , Ouro/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Lectinas/uso terapêutico , Nanopartículas Metálicas/química , Sondas Moleculares/uso terapêutico , Polissacarídeos/uso terapêutico , Sítios de Ligação , Carboidratos/química , Ouro/química , Humanos , Lectinas/química , Ligantes , Sondas Moleculares/síntese química , Sondas Moleculares/química , Estrutura Molecular , Polissacarídeos/químicaRESUMO
The folate-coupled metabolic enzyme MTHFD2 (the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase) confers redox homeostasis and drives cancer cell proliferation and migration. Here, we show that MTHFD2 is hyperacetylated and lysine 88 is the critical acetylated site. SIRT3, the major deacetylase in mitochondria, is responsible for MTHFD2 deacetylation. Interestingly, chemotherapeutic agent cisplatin inhibits expression of SIRT3 to induce acetylation of MTHFD2 in colorectal cancer cells. Cisplatin-induced acetylated K88 MTHFD2 is sufficient to inhibit its enzymatic activity and downregulate NADPH levels in colorectal cancer cells. Ac-K88-MTHFD2 is significantly decreased in human colorectal cancer samples and is inversely correlated with the upregulated expression of SIRT3. Our findings reveal an unknown regulation axis of cisplatin-SIRT3-MTHFD2 in redox homeostasis and suggest a potential therapeutic strategy for cancer treatments by targeting MTHFD2.
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Cisplatino/metabolismo , Neoplasias Colorretais/metabolismo , Sirtuína 3/metabolismo , Acetilação , Aminoidrolases/genética , Aminoidrolases/metabolismo , Aminoidrolases/fisiologia , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/tratamento farmacológico , Ácido Fólico/metabolismo , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Hidrolases , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Metilenotetra-Hidrofolato Desidrogenase (NADP)/fisiologia , Mitocôndrias/metabolismo , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/fisiologia , OxirreduçãoRESUMO
A sulfated fucan was extracted, purified, and characterized from Acaudina leucoprocta (a low value sea cucumber) to better understand and utilize this species. The structure of the sulfated fucan was elucidated using chemical and modern spectroscopic analyses including HPGPC, IR, AFM, GC-MS, and NMR, and its bioactivity was investigated. Our results showed that the sulfated fucan was mainly composed of â 3)-α-L-Fucp-(1â linkage, and that the sulfate groups were substituted at the O-2 and/or O-4 positions of the fucose ring. In detail, the sulfated fucan consisted of Fuc0S (40%), Fuc2S4S (24%), Fuc2S (24%), and Fuc4S (12%). On average, there were seven sulfate groups on every eight fucose residues. Assay for anticoagulant activity indicated that the sulfated fucan displayed intrinsic anticoagulant activity and specific anti-thrombin activity through heparin cofactor II. Our results showed that this bioactive sulfated fucan could enable the high-value utilization of this low-value sea cucumber.
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Anticoagulantes/isolamento & purificação , Polissacarídeos/isolamento & purificação , Animais , Anticoagulantes/química , Anticoagulantes/farmacologia , Testes de Coagulação Sanguínea , Configuração de Carboidratos , Cromatografia por Troca Iônica , Cromatografia Gasosa-Espectrometria de Massas , Cofator II da Heparina/farmacologia , Humanos , Microscopia de Força Atômica , Estrutura Molecular , Peso Molecular , Polissacarídeos/química , Polissacarídeos/farmacologia , Pepinos-do-Mar/química , Análise Espectral , Sulfatos/análiseRESUMO
Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidly in vivo following intramuscular tongue injection by 'click' chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detected in vivo in the brainstem by light microscopy and electron microscopy via silver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using 'click' chemistry.
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Azidas/administração & dosagem , Compostos de Cádmio/administração & dosagem , Toxina da Cólera/administração & dosagem , Neurônios Motores/metabolismo , Nanopartículas/administração & dosagem , Compostos de Selênio/administração & dosagem , Sulfetos/administração & dosagem , Compostos de Zinco/administração & dosagem , Animais , Azidas/química , Tronco Encefálico/metabolismo , Compostos de Cádmio/química , Toxina da Cólera/química , Camundongos , Nanopartículas/química , Compostos de Selênio/química , Sulfetos/química , Compostos de Zinco/químicaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Shiraia bambusicola is a parasitic fungus on the twigs of bamboos. Its relatively large stroma has high medicinal value and can treat a variety of diseases such as rheumatoid arthritis, cold stomach pain, sciatica, injuries, chronic bronchitis, and infantile. It is widely distributed in many provinces in Southern China and also is also found in Japan. AIM OF THE STUDY: Medicinal fungi were important resources for bioactive polysaccharides. To explore bioactive polysaccharides from Shiraia bambusicola, a heteropolysaccharide SB2-1 was purified and obtained from S. bambusicola and its immunostimulating activity was researched. MATERIALS AND METHODS: The polysaccharide from S. bambusicola was extracted and purified using enzyme assisted extraction, ethanol precipitation, anion-exchange and size-exclusion chromatography. Molecular weight of polysaccharide was estimated by high performance gel permeation chromatography. Monosaccharide compositions were determined by high performance liquid chromatography after pre-column derivatization and UV detection. Structure information was elucidated by IR spectrum, GC-MS analysis after methylation and gradual acid hydrolysis of the polysaccharide. The RAW264.7 cells were used to study the immunostimulating activity in vitro. RESULTS: Physicochemical and structural analyses showed that SB2-1 was a neutral heteropolysaccharide with molecular weight at 22.2 kDa and consisted of glucose, galactose and mannose at a ratio of 2.0:1.5:1.0. The structure of SB2-1 was a branched polysaccharides composed of a mannan core and side chains consisted of glucose and galactose. The mannan core was composed of (1â2)-Manp as the main chain. Glucose with (1â4)-D-Glcp, (1â2)-D-Glcp and (1â6)-D-Glcp at different degrees of polymerization were linked at C-6 and C-3 of the (1â2)-Manp as the side chains. The galactose with the linages of (1â6)-D-Galf, â2)-D-Galf(1â and terminal D-Galf(1â also existed in the side chain. The study on the immunostimulating activities of SB2-1 and its core structure P-2 were investigated on RAW264.7 macrophages. The results showed that SB2-1 could activate RAW264.7 macrophage and significantly improve its phagocytic ability by neutral red uptake experiment. Meanwhile, SB2-1 increased significantly higher inducible nitric oxide synthase (iNOS) production and the productions of IL-1, IL-6, IL-12 and TNF-α. The effect of SB2-1 was better than its core structure P-2 produced by gradual acid hydrolysis, which meant the side chains played an important role in the immunostimulating activities. CONCLUSIONS: The investigation demonstrated that the galactofuranose-containing mannogalactoglucan was characteristic polysaccharides in S. bambusicola and could enhance the activation of macrophages.
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Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Ascomicetos/química , Polissacarídeos Fúngicos/química , Polissacarídeos Fúngicos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Camundongos , Células RAW 264.7RESUMO
Full water-dispersion of commercial hydrophobic CdSe/CdS core/shell quantum rods (QRs) was achieved by cap-exchange using a dihydrolipoic acid zwitterion ligand at a low ligand:QR molar ratio (LQMR) of 1000. However, this process almost completely quenched the QR fluorescence, greatly limiting its potential in downstream fluorescence based applications. Fortunately, we found that the QR fluorescence could be recovered by exposure to near ultra-violet to blue light radiation (e.g. 300-450 nm). These "reborn" QRs were found to be compact, bright, and stable, and were resistant to non-specific adsorption, which make them powerful fluorescent probes in broad biomedical applications. We demonstrated their potential in two model applications: first, the QRs were conjugated with His8-tagged small antibody mimetic proteins (also known as Affimers) for the sensitive detection of target proteins via a Förster resonance energy transfer (FRET) readout strategy and second, the QR surface was functionalized with biotins for targeted imaging of cancer cells.
Assuntos
Técnicas Biossensoriais/métodos , Compostos de Cádmio/química , Microscopia de Fluorescência/métodos , Pontos Quânticos/química , Compostos de Selênio/química , Sulfetos/química , Biotina/química , Linhagem Celular Tumoral , Transferência Ressonante de Energia de Fluorescência , Humanos , Ligantes , Luz , Fótons , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Ácido Tióctico/análogos & derivados , Ácido Tióctico/química , Raios UltravioletaRESUMO
A simple magnetic nanoparticle (MNP)-poly-enzyme nanobead sandwich assay for direct detection of ultralow levels of unlabeled target-DNA is developed. This approach uses a capture-DNA covalently linked to a dense PEGylated polymer encapsulated MNP and a biotinylated signal-DNA to sandwich the target-DNA. A DNA ligation is then followed to offer high discrimination between the perfect-match and single-base mismatch target-DNAs. Only the presence of a perfect-match target can covalently link the biotinylated signal-DNA onto the MNP surface for subsequent binding to a polymer nanobead tagged with thousands of copies of high-activity neutravidin-horseradish peroxidase (NAV-HRP) for great enzymatic signal amplification. Combining the advantages of the dense MNP surface PEGylation to reduce non-specific adsorption (assay background) and the powerful signal amplification of poly-enzyme nanobead, this assay can directly quantify the target-DNA down to single digit attomolar with a large linear dynamic range of 5 orders of magnitude (from 10-18 to 10-13M).