RESUMEN
Alzheimer's Disease (AD) is a neuroinflammatory disease characterized partly by the inability to clear, and subsequent build-up, of amyloid-beta (Aß). AD has a bi-directional relationship with circadian disruption (CD) with sleep disturbances starting years before disease onset. However, the molecular mechanism underlying the relationship of CD and AD has not been elucidated. Myeloid-based phagocytosis, a key component in the metabolism of Aß, is circadianly-regulated, presenting a potential link between CD and AD. In this work, we revealed that the phagocytosis of Aß42 undergoes a daily circadian oscillation. We found the circadian timing of global heparan sulfate proteoglycan (HSPG) biosynthesis was the molecular timer for the clock-controlled phagocytosis of Aß and that both HSPG binding and aggregation may play a role in this oscillation. These data highlight that circadian regulation in immune cells may play a role in the intricate relationship between the circadian clock and AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Ritmo Circadiano/fisiología , Proteoglicanos de Heparán Sulfato/metabolismo , Fagocitosis/fisiología , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Relojes Circadianos , Modelos Animales de Enfermedad , Proteoglicanos de Heparán Sulfato/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Agregación Patológica de Proteínas/metabolismoRESUMEN
Creating dual-mode patterns in the same area of the material is an advanced method to increase the dimension of information storage, improve the level of encryption security, and promote the development of encoding technology. However, in situ, different patterns may lead to serious mutual interference in the process of manufacturing and usage. New materials and patterning techniques are essential for the advancement of noninterfering dual-mode patterns. Herein, noninterfering dual-mode patterns are demonstrated by combining the structural color and chromatic polarization, which is designed with an azobenzene-containing linear liquid crystal copolymer featuring a photofluidization effect. On the one hand, structural color patterns are imprinted via silicon templates with periodic microstructures after a UV-light-induced local transition of the polymer surface from a glassy to rubbery state. On the other hand, different polarization patterns based on the local photoinduced orientation of mesogens are created within the photofluidized region by the Weigert effect. Especially, the secondary imprinting is used to eliminate the partial damage to the structural color patterns during writing of the polarization patterns, thus obtaining dual-mode patterns without interference. This study provides a blueprint for the creation of advanced materials and sophisticated photopatterning techniques with potential cross-industry applications.
RESUMEN
BACKGROUND: Camptothecin (CPT), a pentacyclic alkaloid with antitumor properties, is derived from the Camptotheca acuminata. Topotecan and irinotecan (CPT derivatives) were first approved by the Food and Drug Administration for cancer treatment over 25 years ago and remain key anticancer drugs today. However, their use is often limited by clinical toxicity. Despite extensive development efforts, many of these derivatives have not succeeded clinically, particularly in their effectiveness against pancreatic cancer which remains modest. AIM OF THE STUDY: This study aimed to evaluate the therapeutic activity of FLQY2, a CPT derivative synthesized in our laboratory, against pancreatic cancer, comparing its efficacy and mechanism of action with those of established clinical drugs. METHODS: The cytotoxic effects of FLQY2 on cancer cells were assessed using an MTT assay. Patient-derived organoid (PDO) models were employed to compare the sensitivity of FLQY2 to existing clinical drugs across various cancers. The impact of FLQY2 on apoptosis and cell cycle arrest in Mia Paca-2 pancreatic cancer cells was examined through flow cytometry. Transcriptomic and proteomic analyses were conducted to explore the underlying mechanisms of FLQY2's antitumor activity. Western blotting was used to determine the levels of proteins regulated by FLQY2. Additionally, the antitumor efficacy of FLQY2 in vivo was evaluated in a pancreatic cancer xenograft model. RESULTS: FLQY2 demonstrated (1) potent cytotoxicity; (2) superior tumor-suppressive activity in PDO models compared to current clinical drugs such as gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infinitinib, and lenvatinib; (3) significantly greater tumor inhibition than paclitaxel liposomes in a pancreatic cancer xenograft model; (4) robust antitumor effects, closely associated with the inhibition of the TOP I and PDK1/AKT/mTOR signaling pathways. In vitro studies revealed that FLQY2 inhibited cell proliferation, colony formation, induced apoptosis, and caused cell cycle arrest at nanomolar concentrations. Furthermore, the combination of FLQY2 and gemcitabine exhibited significant inhibitory and synergistic effects. CONCLUSION: The study confirmed the involvement of topoisomerase I and the PDK1/AKT/mTOR pathways in mediating the antitumor activity of FLQY2 in treating Mia Paca-2 pancreatic cancer. Therefore, FLQY2 has potential as a novel therapeutic option for patients with pancreatic cancer.
Asunto(s)
Antineoplásicos , Apoptosis , Camptotecina , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas c-akt , Serina-Treonina Quinasas TOR , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Camptotecina/farmacología , Camptotecina/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Proliferación Celular/efectos de los fármacos , Animales , Ratones , Apoptosis/efectos de los fármacos , Relación Estructura-Actividad , Estructura Molecular , Relación Dosis-Respuesta a Droga , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/antagonistas & inhibidores , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora/metabolismo , Ratones Desnudos , Células Tumorales Cultivadas , Línea Celular TumoralRESUMEN
EcGUS has drawn considerable attention for its role as a target in alleviating serious GIAEs. In this study, a series of 72 (thio)urea derivatives were designed, synthesised, and biologically assayed. The bioassay results revealed that E-9 (IC50 = 2.68 µM) exhibited a promising inhibitory effect on EcGUS, surpassing EcGUS inhibitor D-saccharic acid-1,4-lactone (DSL, IC50 = 45.8 µM). Additionally, the inhibitory kinetic study indicated that E-9 (Ki = 1.64 µM) acted as an uncompetitive inhibitor against EcGUS. The structure-activity relationship revealed that introducing an electron-withdrawing group into the benzene ring at the para-position is beneficial for enhancing inhibitory activity against EcGUS. Furthermore, molecular docking analysis indicated that E-9 has a strong affinity to EcGUS by forming interactions with residues Asp 163, Tyr 472, and Glu 504. Overall, these results suggested that E-9 could be a potent EcGUS inhibitor, providing valuable insights and guidelines for the development of future inhibitors targeting EcGUS.
Asunto(s)
Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Inhibidores Enzimáticos , Escherichia coli , Glucuronidasa , Relación Estructura-Actividad , Estructura Molecular , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/metabolismo , Simulación del Acoplamiento Molecular , Tiourea/farmacología , Tiourea/química , Tiourea/síntesis química , GlicoproteínasRESUMEN
Photodeformable liquid crystal polymers (LCPs) exhibit shape changes of different modes like bending, twisting, and oscillation, which depend on the orientation of liquid crystals. However, it is challenging to create a three-dimensional (3D) actuator with distinct actuation modes due to the difficulty of local orientation in a complex bulk architecture. Here we propose a strategy based on athermal photo-welding to integrate different orientations into a single flexible actuator by the photofluidization of azobenzene-containing linear LCPs. Stretch-induced uniaxial films are cut in different directions and subsequently welded via local photofluidization, during which the LCP transitions from a high-modulus glassy state to a rubbery state upon photoisomerization of azobenzene at room temperature. As a consequence, a cucumber vine-like structure with the opposite handedness and a lifting gripper are constructed by such a cut-and-weld process, demonstrating diverse deformation modes of winding, unwinding, and curling. This strategy provides an athermal process for the fabrication of seamless 3D flexible actuators without structural defects, which have potential applications in micromechanical systems, soft robotics, and artificial muscles.
RESUMEN
Kutzneria is a rare genus of Actinobacteria that harbors a variety of secondary metabolite gene clusters and produces several interesting types of bioactive secondary metabolites. Recent efforts have partially elucidated the biosynthetic pathways of some of these bioactive natural products, suggesting the diversity and specificity of secondary metabolism within this genus. Here, we summarized the chemical structures, biosynthetic pathways, and key metabolic enzymes of the secondary metabolites isolated from Kutzneria strains. In-depth comparative genomic analysis of all six available high-quality Kutzneria genomes revealed that the majority (77%) of the biosynthetic gene cluster families of Kutzneria were untapped and identified homologues of key metabolic enzymes in the putative gene clusters, including cytochrome P450s, halogenases, and flavin-dependent N-hydroxylases. The present study suggests that Kutzneria exhibits great potential to synthesize novel secondary metabolites, encodes a variety of valuable metabolic enzymes, and also provides valuable information for the targeted discovery and biosynthesis of novel natural products from Kutzneria.
Asunto(s)
Actinobacteria , Actinomycetales , Productos Biológicos , Metabolismo Secundario , Actinobacteria/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Familia de Multigenes , Productos Biológicos/metabolismo , FilogeniaRESUMEN
α-Glucosidase (AGS) inhibitors have been regarded as an ideal target for the management of type 2 diabetes mellitus (T2DM) since they can maintain an acceptable blood glucose level by delaying the digestion of carbohydrates and diminishing the absorption of monosaccharides. In the process of our endeavor in mining AGS inhibitors from natural sources, the culture broth of two mangrove-derived actinomycetes Streptomyces sp. WHUA03267 and Streptomyces sp. WHUA03072 exhibited an apparent inhibitory activity against AGS. A subsequent chemical investigation into the two extracts furnished 28 secondary metabolites that were identified by spectroscopic methods as two previously undescribed linear polyketides 1-2, four benzenoid ansamycins 3-6, fourteen cyclodipeptides 7-18, one prenylated indole derivative 19, two fusicoccane-type diterpenoids 20-21, two hydroxamate siderophore 22-23, and five others 24-28. Among all of the isolates, 11 and 24 were obtained from actinomycetes for the first time, while 20-21 had never been reported to occur in a marine-derived microorganism previously. In the in vitro AGS inhibitory assay, compounds 3, 8, 9, 11, 14, 16, and 17 exhibited potent to moderate activity with IC50 values ranging from 35.76 ± 0.40 to 164.5 ± 15.5 µM, as compared with acarbose (IC50 = 422.3 ± 8.4 µM). The AGS inhibitory activity of 3, 9, 14, 16, and 17 was reported for the first time. In particular, autolytimycin (3) represented the first ansamycin derivative reported to possess the AGS inhibitory activity. Kinetics analysis and molecular docking were performed to determine the inhibition types and binding modes of these inhibitors, respectively. In the MTT assay, 3, 8, 9, 11, 14, 16, and 17 exhibited no apparent cytotoxicity to the human normal hepatocyte (LO2) cells, suggesting satisfactory safety of these AGS inhibitors.
Asunto(s)
Actinobacteria , Diabetes Mellitus Tipo 2 , Streptomyces , Humanos , Inhibidores de Glicósido Hidrolasas/química , Actinobacteria/metabolismo , Actinomyces/metabolismo , Simulación del Acoplamiento Molecular , Streptomyces/metabolismo , alfa-Glucosidasas/metabolismo , Estructura MolecularRESUMEN
Post-polymerization modification (PPM) offers a versatile approach for engineering multifunctional polymers, but this advantage has not been fully exploited to fabricate multifunctional liquid crystal polymers (LCPs). Here, we design a facile synthetic approach towards multifunctional LCP by combining the ring-opening metathesis polymerization (ROMP) with PPM, in which ROMP helps to prepare a reactive LCP precursor with high molecular weight, and PPM provides a facilitation to introduce functional groups into the precursor. Consequently, a photo- and humidity-responsive linear LCP (LLCP) is demonstrated to show the potential of this synthetic strategy to diversify functions of the LCPs. Under light irradiation and humidity changes, the deformation modes of the LLCP films are converted to complex shapes (bending, twisting, and curling). The obtained dual-responsive LLCP with high molecular weight possesses excellent processability and recyclability, making it possible to construct 3D shape actuators with programmable deformation behaviors under light/humidity.
RESUMEN
Inspired by the action and healing process from living organisms, developing deployable devices using stimuli-responsive materials, or "smart" deployable devices, is desired to realize remote-controlled programmable deformation with additional in situ repair to perform multiple tasks while extending their service life in aerospace. In this work, a photoorganizable triple shape memory polymer (POTSMP) is reported, which is composed of an azobenzene-containing thermoplastic polyurethane. Upon UV and visible illumination, this POTSMP performs arbitrary programming of two temporary shapes and precise and stepwise shape recovery, exhibiting various temporary shapes adapted to different aerospace applications. On the other hand, rapid light-reconfiguration in seconds, including light-reshaping and light-welding, is achieved in response to UV irradiation, allowing in situ localized process and repair of permanent shape. Combining these photoorganizable operations, deformable devices with complex 2D/3D structures are facilely manufactured with no need of special molds. It is envisioned that this POTSMP can expand the potential of photoresponsive TSMPs in smart deployable devices.
Asunto(s)
Materiales Inteligentes , Polímeros/químicaRESUMEN
The manipulation of small amounts of liquids has applications ranging from biomedical devices to liquid transfer. Direct light-driven manipulation of liquids, especially when triggered by light-induced capillary forces, is of particular interest because light can provide contactless spatial and temporal control. However, existing light-driven technologies suffer from an inherent limitation in that liquid motion is strongly resisted by the effect of contact-line pinning. Here we report a strategy to manipulate fluid slugs by photo-induced asymmetric deformation of tubular microactuators, which induces capillary forces for liquid propulsion. Microactuators with various shapes (straight, 'Y'-shaped, serpentine and helical) are fabricated from a mechanically robust linear liquid crystal polymer. These microactuators are able to exert photocontrol of a wide diversity of liquids over a long distance with controllable velocity and direction, and hence to mix multiphase liquids, to combine liquids and even to make liquids run uphill. We anticipate that this photodeformable microactuator will find use in micro-reactors, in laboratory-on-a-chip settings and in micro-optomechanical systems.
RESUMEN
Irinotecan and Topotecan are two Camptothecin derivatives (CPTs) whose resistance is associated with the high expression of breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). To reverse this resistance, two novel CPTs, FL77-28 (7-(3-Fluoro-4-methylphenyl)-10,11-methylenedioxy-20(S)-CPT) and FL77-29 (7-(4-Fluoro-3-methylphenyl)-10,11-methylenedioxy-20(S)-CPT), were synthesized by our group. In this study, the anti-tumor activities of FL77-28, FL77-29, and their parent, FL118 (10,11-methylenedioxy-20(S)-CPT), were evaluated and the results showed that FL77-28 and FL77-29 had stronger anti-tumor activities than FL118. The transport and uptake of FL118, FL77-28, and FL77-29 were investigated in Caco-2 cells for the preliminary prediction of intestinal absorption. The apparent permeability coefficient from apical to basolateral (Papp AP-BL) values of FL77-28 and FL77-29 were (2.32 ± 0.04) × 10-6 cm/s and (2.48 ± 0.18) × 10-6 cm/s, respectively, suggesting that the compounds had moderate absorption. Since the transport property of FL77-28 was passive diffusion and the efflux ratio (ER) was less than 2, two chemical inhibitors were added to further confirm the involvement of efflux proteins. The results showed that FL77-28 was not a substrate of P-gp or BCRP, but FL77-29 was mediated by P-gp. In conclusion, FL77-28 might be a promising candidate to overcome drug resistance induced by multiple efflux proteins.
Asunto(s)
Camptotecina , Proteínas de Neoplasias , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transporte Biológico , Células CACO-2 , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Humanos , Proteínas de Neoplasias/metabolismoRESUMEN
Bacterial secondary metabolites are rich sources of novel drug leads. The diversity of secondary metabolite biosynthetic gene clusters (BGCs) in genome-sequenced bacteria, which will provide crucial information for the efficient discovery of novel natural products, has not been systematically investigated. Here, the distribution and genetic diversity of BGCs in 10 121 prokaryotic genomes (across 68 phyla) were obtained from their PRISM4 outputs using a custom python script. A total of 18 043 BGCs are detected from 5743 genomes with non-ribosomal peptide synthetases (25.4%) and polyketides (15.9%) as the dominant classes of BGCs. Bacterial strains harbouring the largest number of BGCs are revealed and BGC count in strains of some genera vary greatly, suggesting the necessity of individually evaluating the secondary metabolism potential. Additional analysis against 102 strains of discovered bacterial genera with abundant amounts of BGCs confirms that Kutzneria, Kibdelosporangium, Moorea, Saccharothrix, Cystobacter, Archangium, Actinosynnema, Kitasatospora, and Nocardia, may also be important sources of natural products and worthy of priority investigation. Comparative analysis of BGCs within these genera indicates the great diversity and novelty of the BGCs. This study presents an atlas of bacterial secondary metabolite BGCs that provides a lot of key information for the targeted discovery of novel natural products.
Asunto(s)
Vías Biosintéticas , Cianobacterias , Familia de Multigenes , Vías Biosintéticas/genética , Cianobacterias/genética , Metabolismo Secundario/genéticaRESUMEN
BACKGROUND: The formation of supernumerary teeth is an excellent model for studying the molecular mechanisms that control stem/progenitor cell homeostasis needed to generate a renewable source of replacement cells and tissues. Although multiple growth factors and transcriptional factors have been associated with supernumerary tooth formation, the regulatory inputs of extracellular matrix in this regenerative process remains poorly understood. RESULTS: In this study, we present evidence that disrupting glycosaminoglycans (GAGs) in the dental epithelium of mice by inactivating FAM20B, a xylose kinase essential for GAG assembly, leads to supernumerary tooth formation in a pattern reminiscent of replacement teeth. The dental epithelial GAGs confine murine tooth number by restricting the homeostasis of Sox2(+) dental epithelial stem/progenitor cells in a non-autonomous manner. FAM20B-catalyzed GAGs regulate the cell fate of dental lamina by restricting FGFR2b signaling at the initial stage of tooth development to maintain a subtle balance between the renewal and differentiation of Sox2(+) cells. At the later cap stage, WNT signaling functions as a relay cue to facilitate the supernumerary tooth formation. CONCLUSIONS: The novel mechanism we have characterized through which GAGs control the tooth number in mice may also be more broadly relevant for potentiating signaling interactions in other tissues during development and tissue homeostasis.
Asunto(s)
Glicosaminoglicanos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Diente Supernumerario/genética , Animales , Diferenciación Celular , Ratones , Odontogénesis , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Células Madre/metabolismoRESUMEN
Heparin is a widely used biotherapeutic produced from animal tissues. However, it might be possible to produce a bioengineered version using a multienzyme process, relying on the isolation of the E. coli K5 capsule heparosan and its chemical conversion to N-sulfoheparosan, NSH. Glucuronyl C5-epimerase, the first enzyme that acts on NSH, catalyzes the reversible conversion of glucuronic acid (GlcA) to iduronic acid (IdoA). Using full-length NSH, containing different amounts of N-acetylglucosamine (GlcNAc) residues, we demonstrate that C5-epimerase specificity relates to polysaccharide sequence, particularly the location of GlcNAc residues within the chain. We leveraged the deuterium exchange and the novel ß-glucuronidase heparanase BP, which cleaves at the GlcA residue. Liquid chromatography-mass spectrometry and gel permeation chromatography of partial/complete heparanase BP digestion products from various NSH substrates treated with C5-epimerase provide information on C5-epimerase activity and action pattern. This study provides insight into optimizing the large-scale production of bioengineered heparin.
Asunto(s)
Carbohidrato Epimerasas/química , Carbohidrato Epimerasas/metabolismo , Escherichia coli/enzimología , Ácido Glucurónico/química , Polisacáridos/química , Acetilglucosamina/química , Catálisis , Disacáridos/química , Escherichia coli/aislamiento & purificación , Heparina/química , Humanos , Espectrometría de Masas/métodos , Especificidad por SustratoRESUMEN
Urinary glycosaminoglycans (GAGs) can reflect the health condition of a human being, and the GAGs composition can be directly related to various diseases. In order to effectively utilize such information, a detailed understanding of urinary GAGs in healthy individuals can provide insight into the levels and structures of human urinary GAGs. In this study, urinary GAGs were collected and purified from healthy males and females of adults and young adults. The total creatinine-normalized urinary GAG content, molecular weight distribution and disaccharide compositions were determined. Using capillary zone electrophoresis (CZE)-mass spectrometry (MS) and CZE-MS/MS relying on negative electron transfer dissociation, the major components of healthy human urinary GAGs were determined. The structures of 10 GAG oligosaccharides representing the majority of human urinary GAGs were determined.
Asunto(s)
Glicosaminoglicanos/orina , Adulto , Conformación de Carbohidratos , Electroforesis Capilar , Femenino , Voluntarios Sanos , Humanos , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Adulto JovenRESUMEN
A liquid crystal elastomer (LCE) film is successfully deposited with a terahertz metamaterial using thermal evaporation via a programmed electronic shutter and high-efficiency cooling system. The transmittance of the metamaterial at its resonance frequency is monotonically increased from 0.0036 to 1.0 as a pump beam bends the LCE film, so the metamaterial has a large switching contrast of 277 at the frequency. The monotonic increase in the resonance transmittance arises from the constant resonance frequency of the metamaterial at the transmittance modulation and depicts that the metamaterial-deposited LCE film can continuously tune the transmitted intensity of a terahertz beam. The metamaterial-deposited LCE film has potential in developing continuously tunable intensity modulators with large switching contrasts for the application of terahertz imaging and terahertz communication. Therefore, the thermal evaporation expands the application of metamaterials and improves their optical properties.
RESUMEN
Photoresponsive materials offer local, temporal, and remote control over their chemical or physical properties under external stimuli, giving new tools for interfacial regulation. Among all, photodeformable azobenzene-containing liquid crystal polymers (azo-LCPs) have received increasing attention because they can be processed into various micro/nanostructures and have the potential to reversibly tune the interfacial properties through chemical and/or morphological variation by light, providing effective dynamic interface regulation. In this feature article, we highlight the milestones in the dynamic regulation of different interfacial properties through micro/nanostructures made of photodeformable azobenzene-containing liquid crystal polymers (azo-LCPs). We describe the preparation of different azo-LCP micro/nanostructures from the aspects of materials and processing techniques and reveal the importance of mesogen orientation toward dynamic interfacial regulation. By introducing our recently developed linear azo-LCP (azo-LLCP) with good mechanical and photoresponsive performances, we discuss the challenge and opportunity with respect to the dynamic light regulation of two- and three-dimensional (2D/3D) micro/nanostructures to tune their related interfacial properties. We have also given our expectation toward exploring photodeformable micro/nanostructures for advanced applications such as in microfluidics, biosensors, and nanotherapeutics.
RESUMEN
Zika virus (ZIKV) is an enveloped RNA virus from the flavivirus family that can cause fetal neural abnormalities in pregnant women. Previously, we established that ZIKV-EP (envelope protein) binds to human placental chondroitin sulfate (CS), suggesting that CS may be a potential host cell surface receptor in ZIKV pathogenesis. In this study, we further characterized the GAG disaccharide composition of other biological tissues (i.e., mosquitoes, fetal brain cells, and eye tissues) in ZIKV pathogenesis to investigate the role of tissue specific GAGs. Heparan sulfate (HS) was the major GAG, and levels of HS-6-sulfo, HS 0S (unsulfated HS), and CS 4S disaccharides were the main differences in the GAG composition of Aedes aegypti and Aedes albopictus mosquitoes. In human fetal neural progenitor and differentiated cells, HS 0S and CS 4S were the main disaccharides. A change in disaccharide composition levels was observed between undifferentiated and differentiated cells. In different regions of the bovine eyes, CS was the major GAG, and the amounts of hyaluronic acid or keratan sulfate varied depending on the region of the eye. Next, we examined heparin (HP) of various structures to investigate their potential in vitro antiviral activity against ZIKV and Dengue virus (DENV) infection in Vero cells. All compounds effectively inhibited DENV replication; however, they surprisingly promoted ZIKV replication. HP of longer chain lengths more strongly promoted activity in ZIKV replication. This study further expands our understanding of role of GAGs in ZIKV pathogenesis and carbohydrate-based antivirals against flaviviral infection.
Asunto(s)
Aedes/metabolismo , Dengue/tratamiento farmacológico , Ojo/metabolismo , Feto/metabolismo , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/farmacología , Infección por el Virus Zika/tratamiento farmacológico , Aedes/virología , Animales , Antivirales/farmacología , Bovinos , Chlorocebus aethiops , Dengue/metabolismo , Dengue/patología , Dengue/virología , Virus del Dengue/patogenicidad , Ojo/efectos de los fármacos , Feto/efectos de los fármacos , Glicosaminoglicanos/química , Heparitina Sulfato/química , Humanos , Técnicas In Vitro , Mosquitos Vectores/virología , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células Vero , Internalización del Virus , Replicación Viral , Virus Zika/patogenicidad , Infección por el Virus Zika/metabolismo , Infección por el Virus Zika/patología , Infección por el Virus Zika/virologíaRESUMEN
The specificity and action pattern of a ß-glucuronidase derived from the pathogenic bacteria Burkholderia pseudomallei and expressed in Escherichia coli as a recombinant protein has been evaluated. While this enzyme shows activity on a number of glycosaminoglycans, our study has focused on its action on heparin, heparan sulfate and their biosynthetic intermediates as well as chemoenzymatically synthesized, structurally defined heparan sulfate oligosaccharides. These heparin/heparan sulfate (HP/HS) substrates examined varied in size and structure, but all contained an uronic acid (UA) residue ß-(1â4) linked to a glucosamine residue. On the substrates tested, this enzyme (heparanase Bp) acted only on a glucuronic acid residue ß-(1â4) linked to an N-acetylglucosamine, N-sulfoglucosamine or N-acetyl-6-O-sulfoglucosamine residue. A substrate was required to have a length of pentasaccharide or longer and heparanase Bp acted with a random endolytic action pattern on HP/HS. The specificity and glycohydrolase mechanism of action of heparanase Bp resembles mammalian heparanase and is complementary to the bacterial heparin lyases, which act through an eliminase mechanism on a glucosamine residue (1â4) linked to a UA residue, suggesting its utility as a tool for the structural determination of HP/HS as well as representing a possible model for the medically relevant mammalian heparanase. The utility heparanase Bp was demonstrated by the oligosaccharide mapping of heparin, which afforded resistant intact highly sulfated domains ranging from tetrasaccharide to >28-mer with a molecular weight >9000.
Asunto(s)
Proteínas Bacterianas/metabolismo , Burkholderia pseudomallei/enzimología , Glucuronidasa/metabolismo , Heparina/análogos & derivados , Heparina/metabolismo , Heparitina Sulfato/análogos & derivados , Heparitina Sulfato/química , Especificidad por SustratoRESUMEN
Fucosylated chondroitin sulfates (FCSs) from sea cucumbers have repetitive structures that exhibit minor structural differences based on the organism from which they are recovered. A detailed characterization of FCSs and their derivatives is important to establish their structure-activity relationship in the development of new anticoagulant drugs. In the current study, online hydrophilic interaction chromatography-Fourier transform mass spectrometry (FTMS) was applied to analyze the FCS oligosaccharides generated by selective degradation from four species of sea cucumbers, Isostichopus badionotus, Pearsonothuria graeffei, Holothuria mexicana and Acaudina molpadioides. These depolymerized FCS fragments were quantified and compared using the glycomics software package, GlycReSoft. The quantified fragments mainly had trisaccharide-repeating compositions and showed significant differences in fucosylation (including its sulfation) among different species of sea cucumbers. Detailed analysis of FTMS ion peaks and top-down nuclear magnetic resonance spectroscopy of native FCS polysaccharides verified the accuracy of this method. Thus, a new structural model for FCS chains from these different sea cucumbers was defined. This bottom-up approach provides rich detailed structural analysis and provides quantitative information with high accuracy and reproducibility and should be suitable for the quality control in FCSs as well as their oligosaccharides.