RESUMEN
The widespread use of pesticides poses significant threats to both environmental and human health, primarily due to their potential toxic effects. The study investigated the cardiovascular toxicity of selected pesticides, focusing on their interactions with Toll-like receptor 4 (TLR4), an important part of the innate immune system. Using computational tools such as molecular docking, molecular dynamics (MD) simulations, principal component analysis (PCA), density functional theory (DFT) calculations, and ADME analysis, this study identified C160 as having the lowest binding affinity (-8.2 kcal/mol), followed by C107 and C165 (-8.0 kcal/mol). RMSD, RMSF, Rg, and hydrogen bond metrics indicated the formation of stable complexes between specific pesticides and TLR4. PCA revealed significant structural changes upon ligand binding, affecting stability and flexibility, while DFT calculations provided information about the stability, reactivity, and polarity of the compounds. ADME studies highlighted the solubility, permeability, and metabolic stability of C107, C160, and C165, suggesting their potential for bioavailability and impact on cardiovascular toxicity. C107 and C165 exhibit higher bioactivity scores, indicating favourable absorption, metabolism, and distribution properties. C165 also violated rule where molecular weight is greater than 500 g/mol. Further, DFT and NCI analysis of post MD conformations confirmed the binding of ligands at the binding pocket. The analysis shed light on the molecular mechanisms of pesticide-induced cardiovascular toxicity, aiding in the development of strategies to mitigate their harmful effects on human health.
Asunto(s)
Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Plaguicidas , Unión Proteica , Receptor Toll-Like 4 , Pez Cebra , Animales , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Plaguicidas/química , Plaguicidas/metabolismo , Plaguicidas/toxicidad , Humanos , Enlace de Hidrógeno , Ligandos , Mamíferos/metabolismo , Análisis de Componente Principal , Sitios de UniónRESUMEN
Orf virus (ORFV) is an acute contact, epitheliotropic, zoonotic, and double-stranded DNA virus that causes significant economic losses in the livestock industry. The objective of this study is to design an immunoinformatics-based multi-epitope subunit vaccine against ORFV. Various immunodominant cytotoxic T lymphocytes (CTL), helper T lymphocytes (HTL), and B-cell epitopes from the B2L, F1L, and 080 protein of ORFV were selected and linked by short connectors to construct a multi-epitope subunit vaccine. Immunogenicity was enhanced by adding an adjuvant ß-defensin to the N-terminal of the vaccine using the EAAAK linker. The vaccine exhibited a significant degree of antigenicity and solubility, without allergenicity or toxicity. The 3D formation of the vaccine was subsequently anticipated, improved, and verified. The optimized model exhibited a lower Z-score of -4.33, indicating higher quality. Molecular docking results demonstrated that the vaccine strongly binds to TLR2 and TLR4. Molecular dynamics results indicated that the docked vaccine-TLR complexes were stable. Immune simulation analyses further confirmed that the vaccine can induce a marked increase in IgG and IgM antibody titers, and elevated levels of IFN-γ and IL-2. Finally, the optimized DNA sequence of the vaccine was cloned into the vector pET28a (+) for high expression in the E.coli expression system. Overall, the designed multi-epitope subunit vaccine is highly stable and can induce robust humoral and cellular immunity, making it a promising vaccine candidate against ORFV.
Asunto(s)
Epítopos de Linfocito B , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Virus del Orf , Vacunas de Subunidad , Vacunas Virales , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/genética , Vacunas de Subunidad/química , Animales , Virus del Orf/inmunología , Virus del Orf/genética , Vacunas Virales/inmunología , Vacunas Virales/química , Vacunas Virales/genética , Ratones , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/sangre , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/química , Ectima Contagioso/prevención & control , Ectima Contagioso/inmunología , Ectima Contagioso/virología , Ratones Endogámicos BALB C , Femenino , Linfocitos T Citotóxicos/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunologíaRESUMEN
Herein, we describe a rapid and facile fabrication of electrochemical sensors utilizing two different toll-like receptor (TLR) proteins as biorecognition elements to detect bacterial pathogen associated molecular patterns (PAMPs). Using potential-assisted self-assembly, binary mixtures of 11-mercaptoundecanoic acid (MUA) and 6-mercapto-1-hexanol (MCH), or MUA and an in-house synthesized zwitterionic sulfobetaine thiol (DPS) were assembled on a gold working electrode within 5 minutes, which is >200 times shorter than other TLR sensors' preparation time. Electrochemical methods and X-ray photoelectron microscopy were used to characterize the SAM layers. SAMs composed of the betaine terminated thiol exhibited superior resistance to nonspecific interactions, and were used to develop the TLR sensors. Biosensors containing two individually immobilized TLRs (TLR4 and TLR9) were fabricated on separate MUA-DPS SAM modified Au electrodes (MUA-DPS/Au) and tested for their response towards their respective PAMPs. The changes to electron transfer resistance in EIS of the TLR4/MUA-DPS/Au sensor showed a detection limit of 4 ng mL-1 for E. coli 0157:H7 endotoxin (lipopolysaccharide, LPS) and a dynamic range of up to 1000 ng mL-1. The TLR4-based sensor showed negligible response when tested with LPS spiked human plasma samples, showing no interference from the plasma matrix. The TLR9/MUA-DPS/Au sensor responded linearly up to 350 µg mL-1 bacterial DNA, with a detection limit of 7 µg mL-1. The rapid assembly of the TLR sensors, excellent antifouling properties of the mixed SAM assembly, small size and ease of operation of EIS hold great promise for the development of a portable and automated broad-spectrum pathogen detection and classification tool.
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Técnicas Biosensibles , Técnicas Electroquímicas , Hexanoles , Compuestos de Sulfhidrilo , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/sangre , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Humanos , Hexanoles/química , Receptor Toll-Like 4/química , Oro/química , Ácidos Grasos/química , Electrodos , Receptores Toll-Like/química , Moléculas de Patrón Molecular Asociado a Patógenos/química , Escherichia coli/aislamiento & purificación , Receptor Toll-Like 9RESUMEN
Salmonella subsp. enterica (SE) presents a significant global health challenge in both developed and developing countries. Current SE vaccines have limitations, targeting specific strains and demonstrating moderate efficacy in adults, while also being unsuitable for young children and often unaffordable in regions with lower income levels where the disease is prevalent. To address these challenges, this study employed a computational approach integrating core proteomics, subtractive proteomics, and immunoinformatics to develop a universal SE vaccine and identify potential drug targets. Analysis of the core proteome of 185 SE strains revealed 1964 conserved proteins. Subtractive proteomics identified 9 proteins as potential vaccine candidates and 41 as novel drug targets. Using reverse vaccinology-based immunoinformatics, four multi-epitope-based subunit vaccine constructs (MESVCs) were designed, aiming to stimulate cytotoxic T lymphocyte, helper T lymphocyte, and linear B lymphocyte responses. These constructs underwent comprehensive evaluations for antigenicity, immunogenicity, toxicity, hydropathicity, and physicochemical properties. Predictive modeling, refinement, and validation were conducted to determine the secondary and tertiary structures of the SE-MESVCs, followed by docking studies with MHC-I, MHC-II, and TLR4 receptors. Molecular docking assessments showed favorable binding with all three receptors, with SE-MESVC-4 exhibiting the most promising binding energy. Molecular dynamics simulations confirmed the binding affinity and stability of SE-MESVC-4 with the TLR4/MD2 complex. Additionally, codon optimization and in silico cloning verified the efficient translation and successful expression of SE-MESVC-4 in Escherichia coli (E. coli) str. K12. Subsequent in silico immune simulation evaluated the efficacy of SE-MESVC-4 in triggering an effective immune response. These results suggest that SE-MESVC-4 may induce both humoral and cellular immune responses, making it a potential candidate for an effective SE vaccine. However, further experimental investigations are necessary to validate the immunogenicity and efficacy of SE-MESVC-4, bringing us closer to effectively combating SE infections.
Asunto(s)
Biología Computacional , Simulación del Acoplamiento Molecular , Proteómica , Salmonella enterica , Vacunas de Subunidad , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/química , Salmonella enterica/inmunología , Salmonella enterica/química , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Receptor Toll-Like 4/inmunología , Epítopos/inmunología , Epítopos/química , Humanos , Vacunas contra la Salmonella/inmunología , Vacunas contra la Salmonella/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/química , InmunoinformáticaRESUMEN
Puumala orthohantavirus (PUUV) is an emerging zoonotic virus endemic to Europe and Russia that causes nephropathia epidemica, a mild form of hemorrhagic fever with renal syndrome (HFRS). There are limited options for treatment and diagnosis of orthohantavirus infection, making the search for potential immunogenic candidates crucial. In the present work, various bioinformatics tools were employed to design conserved immunogenic peptides containing multiple epitopes of PUUV nucleocapsid protein. Eleven conserved peptides (90% conservancy) of the PUUV nucleocapsid protein were identified. Three conserved peptides containing multiple T and B cell epitopes were selected using a consensus epitope prediction algorithm. Molecular docking using the HPEP dock server demonstrated strong binding interactions between the epitopes and HLA molecules (ten alleles for each class I and II HLA). Moreover, an analysis of population coverage using the IEDB database revealed that the identified peptides have over 90% average population coverage across six continents. Molecular docking and simulation analysis reveal a stable interaction with peptide constructs of chosen immunogenic peptides and Toll-like receptor-4. These computational analyses demonstrate selected peptides' immunogenic potential, which needs to be validated in different experimental systems.
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Simulación del Acoplamiento Molecular , Proteínas de la Nucleocápside , Péptidos , Virus Puumala , Virus Puumala/inmunología , Virus Puumala/genética , Péptidos/inmunología , Péptidos/química , Humanos , Proteínas de la Nucleocápside/inmunología , Proteínas de la Nucleocápside/química , Proteínas de la Nucleocápside/genética , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/química , Fiebre Hemorrágica con Síndrome Renal/inmunología , Fiebre Hemorrágica con Síndrome Renal/virología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/química , Biología Computacional , Secuencia Conservada , Secuencia de Aminoácidos , Unión ProteicaRESUMEN
Innate immune defense mechanisms against infection and cancer encompass the modulation of pattern recognition receptor (PRR)-mediated inflammation, including upregulation of various transcription factors and the activation of pro-inflammatory pathways important for immune surveillance. Dysfunction of PRRs-mediated signaling has been implicated in cancer and autoimmune diseases, while the overactivation of PRRs-driven responses during infection can lead to devastating consequences such as acute lung injury or sepsis. We used crystal structure-based design to develop immunomodulatory lipopolysaccharide (LPS) mimetics targeting one of the ubiquitous PRRs, Toll-like Receptor 4 (TLR4). Taking advantage of an exo-anomeric conformation and specific molecular shape of synthetic nonreducing ß,ß-diglucosamine, which was investigated by NMR, we developed two sets of lipid A mimicking glycolipids capable of either potently activating innate immune responses or inhibiting pro-inflammatory signaling. Stereoselective 1,1'-glycosylation towards fully orthogonally protected nonreducing GlcNß(1â1')ßGlcN followed by stepwise assembly of differently functionalised phosphorylated glycolipids provided biologically active molecules that were evaluated for their ability to trigger or to inhibit cellular innate immune responses. Two LPS mimetics, identified as potent TLR4-specific inducers of the intracellular signaling pathways, serve as vaccine adjuvant- and immunotherapy candidates, while anionic glycolipids with TLR4-inhibitory potential hold therapeutic promise for the management of acute or chronic inflammation.
Asunto(s)
Lipopolisacáridos , Receptor Toll-Like 4 , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Lipopolisacáridos/farmacología , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Glucolípidos/química , Glucolípidos/farmacologíaRESUMEN
Introduction: Trypanosoma cruzi is a protozoan parasite that causes the tropical ailment known as Chagas disease, which has its origins in South America. Globally, it has a major impact on health and is transported by insect vector that serves as a parasite. Given the scarcity of vaccines and the limited treatment choices, we conducted a comprehensive investigation of core proteomics to explore a potential reverse vaccine candidate with high antigenicity. Methods: To identify the immunodominant epitopes, T. cruzi core proteomics was initially explored. Consequently, the vaccine sequence was engineered to possess characteristics of non-allergenicity, antigenicity, immunogenicity, and enhanced solubility. After modeling the tertiary structure of the human TLR4 receptor, the binding affinities were assessed employing molecular docking and molecular dynamics simulations (MDS). Results: Docking of the final vaccine design with TLR4 receptors revealed substantial hydrogen bond interactions. A server-based methodology for immunological simulation was developed to forecast the effectiveness against antibodies (IgM + IgG) and interferons (IFN-g). The MDS analysis revealed notable levels of structural compactness and binding stability with average RMSD of 5.03 Aring;, beta-factor 1.09e+5 Å, Rg is 44.7 Aring; and RMSF of 49.50 Aring;. This is followed by binding free energies calculation. The system stability was compromised by the complexes, as evidenced by their corresponding Gibbs free energies of -54.6 kcal/mol. Discussion: Subtractive proteomics approach was applied to determine the antigenic regions of the T cruzi. Our study utilized computational techniques to identify B- and T-cell epitopes in the T. cruzi core proteome. In current study the developed vaccine candidate exhibits immunodominant features. Our findings suggest that formulating a vaccine targeting the causative agent of Chagas disease should be the initial step in its development.
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Enfermedad de Chagas , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteoma , Vacunas Antiprotozoos , Receptor Toll-Like 4 , Trypanosoma cruzi , Trypanosoma cruzi/inmunología , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/prevención & control , Humanos , Proteoma/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Vacunas Antiprotozoos/inmunología , Animales , Epítopos Inmunodominantes/inmunología , Proteómica/métodos , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/química , Anticuerpos Antiprotozoarios/inmunología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/química , Desarrollo de Vacunas , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/químicaRESUMEN
Toll-like receptors (TLRs) are major players in the innate immune system-recognizing pathogens and differentiating self/non-self components of immunity. These proteins are present either on the plasma membrane or endosome and recognize pathogens at their extracellular domains. They are characterized by a single transmembrane helix and an intracellular toll-interleukin-1 receptor (TIR) domain. Few TIRs directly invoke downstream signaling, while others require other TIR domains of adaptors like TIR domain-containing adaptor-inducing interferon-ß (TRIF) and TRIF-related adaptor molecule (TRAM). On recognizing pathogenic lipopolysaccharides, TLR4 dimerises and interacts with the intracellular TRAM dimer through the TIR domain to recruit a downstream signaling adaptor (TRIF). We have performed an in-depth study of the structural effect of two mutations (P116H and C117H) at the dimeric interface of the adaptor TRAM, which are known to abrogate downstream signaling. We modeled the structure and performed molecular dynamics studies in order to decipher the structural basis of this effect. We observed that these mutations led to an increased radius of gyration of the complex and resulted in several changes to the interaction energy values when compared against the wild type (WT) and positive control mutants. We identified highly interacting residues as hubs in the WT dimer, and a few such hubs that were lost in the mutant dimers. Changes in the protein residue path, hampering the information flow between the crucial A86/E87/D88/D89 and T155/S156 sites, were observed for the mutants. Overall, we show that such residue changes can have subtle but long-distance effects, impacting the signaling path allosterically.
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Proteínas Adaptadoras del Transporte Vesicular , Simulación de Dinámica Molecular , Multimerización de Proteína , Humanos , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Transducción de Señal , Unión Proteica , Receptor Toll-Like 4/química , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/genética , Mutación , Dominios y Motivos de Interacción de Proteínas , Expresión Génica , Conformación Proteica en Hélice alfa , Sitios de Unión , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
Haemophilus parainfluenzae is a Gram-negative opportunist pathogen within the mucus of the nose and mouth without significant symptoms and has an ability to cause various infections ranging from ear, eye, and sinus to pneumonia. A concerning development is the increasing resistance of H. parainfluenzae to beta-lactam antibiotics, with the potential to cause dental infections or abscesses. The principal objective of this investigation is to utilize bioinformatics and immuno-informatic methodologies in the development of a candidate multi-epitope Vaccine. The investigation focuses on identifying potential epitopes for both B cells (B lymphocytes) and T cells (helper T lymphocytes and cytotoxic T lymphocytes) based on high non-toxic and non-allergenic characteristics. The selection process involves identifying human leukocyte antigen alleles demonstrating strong associations with recognized antigenic and overlapping epitopes. Notably, the chosen alleles aim to provide coverage for 90% of the global population. Multi-epitope constructs were designed by using suitable linker sequences. To enhance the immunological potential, an adjuvant sequence was incorporated using the EAAAK linker. The final vaccine construct, comprising 344 amino acids, was achieved after the addition of adjuvants and linkers. This multi-epitope Vaccine demonstrates notable antigenicity and possesses favorable physiochemical characteristics. The three-dimensional conformation underwent modeling and refinement, validated through in-silico methods. Additionally, a protein-protein molecular docking analysis was conducted to predict effective binding poses between the multi-epitope Vaccine and the Toll-like receptor 4 protein. The Molecular Dynamics (MD) investigation of the docked TLR4-vaccine complex demonstrated consistent stability over the simulation period, primarily attributed to electrostatic energy. The docked complex displayed minimal deformation and enhanced rigidity in the motion of residues during the dynamic simulation. Furthermore, codon translational optimization and computational cloning was performed to ensure the reliability and proper expression of the multi-Epitope Vaccine. It is crucial to emphasize that despite these computational validations, experimental research in the laboratory is imperative to demonstrate the immunogenicity and protective efficacy of the developed vaccine. This would involve practical assessments to ascertain the real-world effectiveness of the multi-epitope Vaccine.
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Biología Computacional , Epítopos de Linfocito B , Epítopos de Linfocito T , Humanos , Epítopos de Linfocito T/inmunología , Biología Computacional/métodos , Epítopos de Linfocito B/inmunología , Simulación del Acoplamiento Molecular , Infecciones por Haemophilus/prevención & control , Infecciones por Haemophilus/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Desarrollo de VacunasRESUMEN
As one innate immune pattern recognition receptor, Toll-like receptor 4 (TLR4) recently has been considered as a critical player in glucolipid metabolism. Blueberries contain high level of anthocyanins, especially malvidin-3-glucoside (Mv-3-glc), which contribute the anti-inflammatory, hypoglycemic, and hypolipidemic effects. It is speculated that Mv-3-glc is able to possess these functions by binding to TLR4. Here, the noncovalent interactions of Mv-3-glc and TLR4 was explored through multi-techniques including fluorescence and ultraviolet-visible (UV-Vis) absorption spectroscopy, as well as molecular docking. The results demonstrated that Mv-3-glc was able to quench TLR4 intrinsic fluorescence effectively. A stable complex was formed spontaneously and the reaction was exothermic. The degree of binding of Mv-3-glc to TLR4 showed a strong dependence on the chemical concentration, temperature, and pH values. The negative signs for enthalpy (ΔH = -69.1 ± 10.8 kJ/mol) and entropy (ΔS = -105.0 ± 12.3 J/mol/K) from the interaction of the Mv-3-glc and TLR4 shows that the major driving forces are the hydrogen bonding and van der Waals' force, which is consistent with the molecular docking results. In addition, molecular docking predicted that the active center with specific amino acid residues, Phe126, Ser127, Leu54, Ile153, and Tyr131 was responsible for the site of Mv-3-glc binding to TLR4/myeloid differentiation protein-2 (MD-2). These findings confirmed that Mv-3-glc could bind to TLR4, which would be beneficial to understand the target therapeutic effects of blueberry anthocyanins on TLR4 in regulating glucolipid metabolism.
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Antocianinas , Glucósidos , Simulación del Acoplamiento Molecular , Espectrometría de Fluorescencia , Receptor Toll-Like 4 , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Glucósidos/química , Glucósidos/metabolismo , Antocianinas/química , Antocianinas/metabolismo , Antocianinas/farmacología , Humanos , Unión Proteica , Espectrofotometría Ultravioleta , Termodinámica , Enlace de Hidrógeno , Sitios de UniónRESUMEN
BACKGROUND: Dry eye disease is the most commonplace multifractional ocular complication, which has already affected millions of people in the world. It is identified by the excessive buildup of reactive oxygen species, leading to substantial corneal epithelial cell demise and ocular surface inflammation attributed to TLR4. In this study, we aimed to identify potential compounds to treat of dry eye syndrome by exploring in silico methods. METHODS: In this research, molecular docking and dynamics simulation tests were used to examine the effects of selected compounds on TLR4 receptor. Compounds were extracted from different databases and were prepared and docked against TLR4 receptor via Autodock Vina. Celastrol, lumacaftor and nilotinib were selected for further molecular dynamics studies for a deeper understanding of molecular systems consisting of protein and ligands by using the Desmond module of the Schrodinger Suite. RESULTS: The docking results revealed that the compounds are having binding affinity in the range of -5.1 to -8.78 based on the binding affinity and three-dimensional interactions celastrol, lumacaftor and nilotinib were further studied for their activity by molecular dynamics. Among the three compounds, celastrol was the most stable based on molecular dynamics trajectory analysis from 100 ns in the catalytic pockets of 2Z63.pdb.pdb. Root mean square deviation of celastrol/2Z63 was in the range of 1.8-4.8 Å. CONCLUSION: In particular, Glu376 of TLR4 receptor is crucial for the identification and binding of lipopolysaccharides (LPS), which are part of Gram-negative bacteria's outer membrane. In our investigation, celastrol binds to Glu376, suggesting that celastrol may prevent the dry eye syndrome by inhibiting LPS's binding to TLR4.
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Síndromes de Ojo Seco , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Triterpenos Pentacíclicos , Pirimidinas , Receptor Toll-Like 4 , Síndromes de Ojo Seco/tratamiento farmacológico , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Humanos , Triterpenos Pentacíclicos/farmacología , Triterpenos Pentacíclicos/química , Triterpenos Pentacíclicos/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/química , Pirimidinas/uso terapéutico , Triterpenos/farmacología , Triterpenos/química , Simulación por Computador , Ligandos , Aminopiridinas/farmacología , Aminopiridinas/química , Aminopiridinas/uso terapéuticoRESUMEN
The purpose of this study was to identify ovalbumin-derived immunomodulatory peptides by in vitro cell experiments, de novo sequencing, and molecular docking. Ovalbumin hydrolysates were prepared by two enzymes (alkaline protease and papain) individually, sequentially, or simultaneously, respectively. The simultaneous enzymatic hydrolysate (OVAH) had a high degree of hydrolysis (38.12 ± 0.48%) and exhibited immune-enhancing and anti-inflammatory activities. A total of 160 peptides were identified by LC-MS/MS in OVAH. Three novel peptides NVMEERKIK, ADQARELINS, and WEKAFKDE bound to TLR4-MD2 through hydrogen bonds and hydrophobic interactions with high binding affinity and binding energies of -181.40, -178.03, and -168.12 kcal/mol, respectively. These three peptides were synthesized and validated for two-way immunomodulatory activity. NVMEERKIK exhibiting the strongest immunomodulatory activity, increased NO and TNF-α levels by 128.69 and 38.01%, respectively, in normal RAW264.7 cells and reduced NO and TNF-α levels by 27.31 and 39.13%, respectively, in lipopolysaccharide-induced inflammatory RAW264.7 cells. Overall, this study first revealed that ovalbumin could be used as an immunomodulatory source for controlling inflammatory factor secretion.
Asunto(s)
Simulación del Acoplamiento Molecular , Ovalbúmina , Péptidos , Ovalbúmina/inmunología , Ovalbúmina/química , Ratones , Animales , Células RAW 264.7 , Péptidos/química , Péptidos/farmacología , Péptidos/inmunología , Receptor Toll-Like 4/química , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/genética , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Agentes Inmunomoduladores/química , Agentes Inmunomoduladores/farmacología , Secuencia de Aminoácidos , Espectrometría de Masas en Tándem , Óxido Nítrico/metabolismo , Óxido Nítrico/inmunología , Factores Inmunológicos/química , Factores Inmunológicos/farmacologíaRESUMEN
Agonists of Toll like receptors (TLRs) have attracted interest as adjuvants and immune modulators. A crystal structure of TLR4/MD2 with E. coli LPS indicates that the fatty acid at C-2 of the lipid A component of LPS induces dimerization of two TLR4-MD2 complexes, which in turn initiates cell signaling leading to the production of (pro)inflammatory cytokines. To probe the importance of the (R)-3-hydroxymyristate at C-2 of lipid A, a range of bis- and mono-phosphoryl lipid A derivatives with different modifications at C-2 were prepared by a strategy in which 2-methylnaphthyl ethers were employed as permanent protecting group that could be readily removed by catalytic hydrogenation. The C-2 amine was protected as 9-fluorenylmethyloxycarbamate, which at a later stage could be removed to give a free amine that was modified by different fatty acids. LPS and the synthetic lipid As induced the same cytokines, however, large differences in activity were observed. A compound having a hexanoyl moiety at C-2 still showed agonistic properties, but further shortening to a butanoyl abolished activity. The modifications had a larger influence on monophosphoryl lipid As. The lipid As having a butanoyl moiety at C-2 could selectively antagonize TRIF associated cytokines induced by LPS or lipid A.
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Citocinas , Lípido A , Lipopolisacáridos , Lípido A/química , Lípido A/farmacología , Lípido A/análogos & derivados , Lípido A/síntesis química , Citocinas/metabolismo , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Humanos , Antígeno 96 de los Linfocitos/metabolismo , Antígeno 96 de los Linfocitos/química , Diseño de Fármacos , Relación Estructura-Actividad , Transducción de Señal/efectos de los fármacosRESUMEN
Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry.
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Lípido A , Lípido A/química , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 4/química , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/química , Animales , Lipopolisacáridos/química , RatonesRESUMEN
S100A8 and S100A9 belong to the calcium-binding, damage associated molecular pattern (DAMP) proteins shown to aggravate the pathogenesis of rheumatoid arthritis (RA) through their interaction with the TLR4, RAGE and CD36 receptors. S100A8 and S100A9 proteins tend to exist in monomeric, homo and heterodimeric forms, which have been implicated in the pathogenesis of RA, via interacting with Pattern Recognition receptors (PRRs). The study aims to assess the influence of changes in the structure and biological assembly of S100A8 and S100A9 proteins as well as their interaction with significant receptors in RA through computational methods and surface plasmon resonance (SPR) analysis. Molecular docking analysis revealed that the S100A9 homodimer and S100A8/A9 heterodimer showed higher binding affinity towards the target receptors. Most S100 proteins showed good binding affinity towards TLR4 compared to other receptors. Based on the 50 ns MD simulations, TLR4, RAGE, and CD36 formed stable complexes with the monomeric and dimeric forms of S100A8 and S100A9 proteins. However, SPR analysis showed that the S100A8/A9 heterodimers formed stable complexes and exhibited high binding affinity towards the receptors. SPR data also indicated that TLR4 and its interactions with S100A8/A9 proteins may play a primary role in the pathogenesis of RA, with additional contributions from CD36 and RAGE interactions. Subsequent in vitro and in vivo investigations are warranted to corroborate the involvement of S100A8/A9 and the expression of TLR4, RAGE, and CD36 in the pathophysiology of RA.
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Antígenos CD36 , Calgranulina A , Calgranulina B , Simulación del Acoplamiento Molecular , Receptor para Productos Finales de Glicación Avanzada , Receptor Toll-Like 4 , Calgranulina B/química , Calgranulina B/metabolismo , Receptor Toll-Like 4/química , Receptor Toll-Like 4/metabolismo , Calgranulina A/química , Calgranulina A/metabolismo , Calgranulina A/genética , Humanos , Antígenos CD36/química , Antígenos CD36/metabolismo , Antígenos CD36/genética , Receptor para Productos Finales de Glicación Avanzada/química , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Unión Proteica , Simulación de Dinámica Molecular , Resonancia por Plasmón de Superficie , Multimerización de Proteína , Artritis Reumatoide/metabolismoRESUMEN
Moraxella lacunata is an emerging gram-negative bacterium that is responsible for multiple nosocomial infections. The bacterium is evolving resistance to several antibiotics, and currently, no effective licensed vaccines are available, which warrants the search for new therapeutics. A multi-epitope-based vaccine has been designed for M. lacunata. The complete proteome of M. lacunata contains 10,110 core proteins. Subcellular localization analysis revealed the presence of five proteins in the extracellular matrix, while 19 proteins were predicted to be located in the outer membrane, and 21 proteins were predicted to be located in the periplasmic region. Only two proteins, the type VI secretion system tube protein (Hcp) and the transporter substrate-binding domain-containing protein, were selected for epitope prediction as they fulfilled all the criteria for being potential vaccine candidates. Shortlisted epitopes from the selected proteins were fused together using "GPGPG" linkers to overcome the limitations of single-epitope vaccines. Next, the cholera toxin-B adjuvant was attached to the peptide epitope using an EAAAK linker. Docking analysis was performed to examine the interaction between the vaccine and immune cell receptors, revealing robust intermolecular interactions and a stable binding conformation. Molecular dynamics simulation findings revealed no drastic changes in the binding conformation of complexes during the simulation period. The net binding free energy of vaccine-receptor complexes was estimated using the molecular mechanics energies combined with the Poisson-Boltzmann and surface area continuum solvation (MM-PBSA) method. The reported values were -586.38 kcal/mol, -283.74 kcal/mol, and -296.88 kcal/mol for the TLR-4-vaccine complex, MHC-I-vaccine complex, and MHC-II-vaccine complex, respectively. Furthermore, the molecular mechanics energies combined with the generalized Born and surface area continuum solvation (MM-GBSA) analysis predicted binding free energies of -596.69 kcal/mol, -287.39 kcal/mol, and -298.28 kcal/mol for the TLR-4-vaccine complex, MHC-I-vaccine complex, and MHC-II-vaccine complex, respectively. The theoretical vaccine design proposed in the study could potentially serve as a powerful therapeutic against targeted pathogens, subject to validation through experimental studies.Communicated by Ramaswamy H. Sarma.
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Simulación de Dinámica Molecular , Moraxella , Receptor Toll-Like 4 , Receptor Toll-Like 4/química , Epítopos , Vacunas Bacterianas , Simulación del Acoplamiento Molecular , Biología Computacional/métodos , Epítopos de Linfocito T , Vacunas de Subunidad , Epítopos de Linfocito BRESUMEN
BACKGROUND: Multidrug-resistant Pseudomonas aeruginosa has become a major cause of severe infections. Due to the lack of approved vaccines, this study has presented putative vaccine candidates against it. METHODS: P. aeruginosa 24Pae112 as a reference strain was retrieved from GenBank database. The surface-exposed, antigenic, non-allergenic, and non-homologous human proteins were selected. The conserved domains of selected proteins were evaluated, and the prevalence of proteins was assessed among 395 genomes. Next, linear and conformational B-cell epitopes, and human MHC II binding sites were determined. Finally, five conserved and highly antigenic B-cell epitopes from OMPs were implanted on the three platforms as multi-epitope vaccines, including FliC, the bacteriophage T7 tail, and the cell wall-associated transporter proteins. The immunoreactivity was investigated using molecular docking and immune simulation. Furthermore, molecular dynamics simulation was done to refine the chimeric cell-wall-associated transporter-TLR4 complex as the best interaction. RESULTS: Among 6494 total proteins of P. aeruginosa 24Pae112, 16 proteins (seven OMPs and nine secreted) were ideal according to the defined criteria. These proteins had a molecular weight of 110 kDa and were prevalent in ≥ 75% of P. aeruginosa genomes. Among the presented multi-epitope vaccines, the chimeric cell-wall-associated transporter had the strongest interaction with TLR4. Moreover, the immune simulation response revealed that the bacteriophage T7 tail chimeric protein had the strongest ability to stimulate the immune system. In addition, molecular docking and molecular dynamic simulation indicated the proper and stable interactions between the chimeric cell-wall-associated transporter and TLR4. CONCLUSION: This study proposed 16 shortlisted proteins as promising immunogenic targets. Two novel platforms (e.g. cell-wall-associated transporter and bacteriophage T7 tail proteins) for designing of multi-epitope vaccines (MEVs), showed the better performance compared to FliC. In our future studies, these two MEVs will receive more scrutiny to evaluate their immunoreactivity.
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Epítopos de Linfocito B , Pseudomonas aeruginosa , Humanos , Simulación del Acoplamiento Molecular , Vacunología , Receptor Toll-Like 4/química , Simulación de Dinámica Molecular , Epítopos de Linfocito T , Biología Computacional , Vacunas de SubunidadRESUMEN
The human toll-like receptor (hTLR) 4 single nucleotide polymorphisms (SNPs) are interconnected with cancer, multiple genetic disorders and other immune-related diseases. The detrimental effect of SNPs in hTLR4 with respect to structure and function has not been explored in depth. The present study concatenates the biological consequences of the SNPs along with structural modifications predicted at the hTLR4 gene. A total of 7910 SNPs of hTLR4 were screened, and 21 damage-causing SNPs were identified. Out of 21, seven are present in the extracellular region, of which three were detected as deleterious and the fourth one as moderate. These three mutations are located in a highly conserved region and influence conformational change. The change leads to the widening of the Leucine-rich repeat (LRR) arc to a maximum of 16.9 Å and a minimum of 8.7 Å. Expansion/shortening of LRR arc, never discussed before, would cause loss of myeloid differentiation factor 2 (MD-2) interactions in the interior and diminish lipopolysaccharide (LPS) responses. Similarly, in all mutant structures, the binding region for HMGB1 and LPS is deflating or in an unsupportive conformation. Thus, SNPs affect the regular signaling cascade and might result in human sepsis, genetic disorders, cancer and other immunological related diseases.Communicated by Ramaswamy H. Sarma.
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Simulación de Dinámica Molecular , Neoplasias , Humanos , Receptor Toll-Like 4/química , Polimorfismo de Nucleótido Simple , Lipopolisacáridos/químicaRESUMEN
OBJECTIVE: To investigate the effects of octreotide and nateglinide on ovarian follicle count, ovarian tissue damage, biochemical parameters and free radical scavenging system in letrazole-induced rat model of PCOS. MATERIALS AND METHODS: Forty-two female Sprague-Dawley rats were divided into six groups. Group 1 (Control Group): after localizing the ovaries and the uterine horns, the abdominal wall was closed without any surgical procedure. Group 2 (PCOS Group): PCOS was induced by administrating Letrozole orally for 21 successive days. At the end of 21 days, rats underwent ovarian biopsies. The experimental PCOS model was considered successful in the presence of atretic follicles without granulosa cell stratification. Group 3 (PCOS + Nateglinide Group): Nateglinide was administered by oral dropper for 30 days to the rats in which PCOS model was created. Group 4 (Nateglinid only Group): 30 days of NG was applied to the rats without PCOS. Group 5 (PCOS+Octreotide Group): 0.1 mg/kg/day Octreotide was given intraperitoneally for 4 weeks to the rats in which PCOS model was created. Group 6 (Octreotide only Group): animals without PCOS given 0.1 mg/kg/day Octreotide at the end of the treatment, bilateral oophorectomy was performed and blood samples were collected from all groups. Ovarian tissue was stained immunohistochemically with TLR-4 in addition to conventional staining. In addition to follicle classification, ovarian damage was graded. Serum insulin, FSH and LH, TNF-α, IL-6, SHBG, SOD, IGF-1, MDA and GSH levels were also measured. RESULTS: The cystic and degenerated follicle density of PCOS group was high compared with the other groups. Both cystic and degenerated follicles were significantly reduced in PCOS+NG and PCOS+OC groups compared to PCOS group. There was no difference between the groups in terms of serum LH, FSH and insulin levels (p>0.05). Serum testosterone level was significantly higher in the PCOS group compared to the other groups (p<0.01). Adding OC or NG to PCOS groups did not cause significant changes in testosterone levels. TNF-α and IL-6 levels were high in PCOS group (p<0.03). IGF-1 and MDA levels were higher in PCOS than in other groups (p<0.03, p<0.01 respectively). Adding OC or NG to the treatment normalized IGF-1 and MDA levels. Serum GSH levels were significantly lower in the PCOS group (p<0.05). Adding NG to the treatment increased GSH levels. CONCLUSIONS: Both NG and OCT reverses atretic and degenerate follicle damage due to PCOS through TLR-4, antioxidant and anti-inflammatory pathways.
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Insulinas , Nateglinida , Octreótido , Síndrome del Ovario Poliquístico , Animales , Femenino , Ratas , Modelos Animales de Enfermedad , Hormona Folículo Estimulante/química , Radicales Libres , Factor I del Crecimiento Similar a la Insulina , Interleucina-6 , Nateglinida/farmacología , Nateglinida/uso terapéutico , Octreótido/farmacología , Octreótido/uso terapéutico , Síndrome del Ovario Poliquístico/inducido químicamente , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Síndrome del Ovario Poliquístico/patología , Ratas Sprague-Dawley , Testosterona , Receptor Toll-Like 4/química , Factor de Necrosis Tumoral alfa/química , Letrozol/farmacologíaRESUMEN
In the current study, full-length Toll-like receptor 4 (TLR4) cDNA was cloned and characterised in Tor putitora, an important fish inhibiting Himalayan rivers. The complete coding sequence of TpTLR4 is 2457 bp with nine key structural domains, including six leucine-rich repeats (LRRs). The phylogenetic tree revealed that TpTLR4 showed the closest relationship with TLR4 of Cyprinus carpio (96%), Labeo rohita (91%) and Megalobrama amblycephala (88%), all belonging to the Cyprinidae family. CELLO2GO tool revealed that TpTLR4 protein is highly localised in the plasma (67.7%), and the protein has a strong association with myeloid differentiation primary response 88 (MYD88) followed by Tumor necrosis factor receptor-associated factor (TRAF) family. In the toll-interleukin-1 receptor (TIR) domain of TpTLR4, the proline is replaced by the alanine amino acid, thus may give plasticity to the receptor to recognise both bacterial and viral ligands. Molecular docking has revealed that TpTLR4 showed the strongest affinity towards poly (I:C) with the binding energy of -6.1 kcal/mol and five hydrogen bonds among all ligands. Based on our molecular docking results, it can be presumed that TpTLR4 can sense bacterial, fungal and viral molecular patterns with binding sites mainly present in the TpTLR4 LRR9 motif, which spans between 515 and 602 amino acids. Tor putiora TLR4 transcript was ubiquitously expressed in all the tested fish tissues. Although, transcript level was found to be highest in blood and spleen followed by the kidney. The TpTLR4 transcripts showed peak expression in spleen and kidney at 12 h post-injection (hpi) (p < 0.05) of poly (I:C). The constitutive expression of TpTLR4 in various tissues, up-regulation in different tissues and strong binding affinities with poly (I:C) indicate that TpTLR4 may play an essential role in sensing pathogen-associated molecular patterns (PAMPs), particularly of viral origin.