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Hematopoietic humanized (hu) mice are powerful tools for modeling the action of human immune system and are widely used for preclinical studies and drug discovery. However, generating a functional human T cell compartment in hu mice remains challenging, primarily due to the species-related differences between human and mouse thymus. While engrafting human fetal thymic tissues can support robust T cell development in hu mice, tissue scarcity and ethical concerns limit their wide use. Here, we describe the tissue engineering of human thymus organoids from inducible pluripotent stem cells (iPSC-thymus) that can support the de novo generation of a diverse population of functional human T cells. T cells of iPSC-thymus-engrafted hu mice could mediate both cellular and humoral immune responses, including mounting robust proinflammatory responses on T cell receptor engagement, inhibiting allogeneic tumor graft growth and facilitating efficient Ig class switching. Our findings indicate that hu mice engrafted with iPSC-thymus can serve as a new animal model to study human T cell-mediated immunity and accelerate the translation of findings from animal studies into the clinic.
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Transplante de Células-Tronco Hematopoéticas , Células-Tronco Pluripotentes Induzidas , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos SCID , Organoides , Linfócitos T , TimoRESUMO
Peptide-based therapeutics have been gaining attention due to their ability to actively target tumor cells. Additionally, several varieties of nucleotide derivatives have been developed to reduce cell proliferation and induce apoptosis of tumor cells. In this work, we have developed novel peptide conjugates with newly designed purine analogs and pyrimidine derivatives and explored the binding interactions with the kinase domain of wild-type EGFR and its mutant EGFR [L858R/ T790M] which are known to be over-expressed in tumor cells. The peptides explored included WNWKV (derived from sea cucumber) and LARFFS, which in previous work was predicted to bind to Domain I of EGFR. Computational studies conducted to explore binding interactions include molecular docking studies, molecular dynamics simulations and MMGBSA to investigate the binding abilities and stability of the complexes. The results indicate that conjugation enhanced binding capabilities, particularly for the WNWKV conjugates. MMGBSA analysis revealed nearly twofold higher binding toward the T790M/L858R double mutant receptor. Several conjugates were shown to have strong and stable binding with both wild-type and mutant EGFR. As a proof of concept, we synthesized pyrimidine conjugates with both peptides and determined the KD values using SPR analysis. The results corroborated with the computational analyses. Additionally, cell viability and apoptosis studies with lung cancer cells expressing the wild-type and double mutant proteins revealed that the WNWKV conjugate showed greater potency than the LARFFS conjugate, while LARFFS peptide alone showed poor binding to the kinase domain. Thus, we have designed peptide conjugates that show potential for further laboratory studies for developing therapeutics for targeting the EGFR receptor and its mutant T790M/L858R.
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Recent studies have revealed that MERTK and BRAF V600E receptors have been found to be over-expressed in several types of cancers including melanoma, making these receptors targets for drug design. In this study, we have designed novel peptide conjugates with the natural products vanillic acid, thiazole-2-carboxylic acid, cinnamic acid, theanine, and protocatechuic acid. Each of these compounds was conjugated with the tumor targeting peptide sequence TAASGVRSMH, known to bind to NG2 and target tumor neovasculature. We examined their binding affinities and stability with MERTK and BRAF V600E receptors using molecular docking and molecular dynamics studies. Compared to the neat compounds, the peptide conjugates displayed higher binding affinity toward both receptors. In the case of MERTK, the most stable complexes were formed with di-theaninate-peptide, vanillate-peptide, and thiazole-2-amido peptide conjugates and binding occurred in the hinge region. Additionally, it was discovered that the peptide alone also had high binding ability and stability with the MERTK receptor. In the case of BRAF V600E, the peptide conjugates of protocatechuate, vanillate and thiazole-2-amido peptide conjugates showed the formation of the most stable complexes and binding occurred in the ATP binding cleft. Further analysis revealed that the number of hydrogen bonds and hydrophobic interactions played a critical role in enhanced stability of the complexes. Docking studies also revealed that binding affinities for NG2 were similar to MERTK and higher for BRAF V600E. MMGBSA studies of the trajectories revealed that the protocatechuate-peptide conjugate showed the highest binding energy with BRAF V600E while the peptide-TAASGVRSMH showed the highest binding energy with MERTK. ADME studies revealed that each of the compounds showed medium to high permeability toward MDCK cells and were not hERG blockers. Furthermore, the conjugates were not CYP inhibitors or substrates, but they were found to be Pgp substrates. Our results indicated that the protocatechuate-TAASGVRSMH, thiazole-2-amido-TAASGVRSMH, and vanillate-TAASGVRSMH conjugates may be furthered developed for in vitro and in vivo studies as novel tumor targeting compounds for tumor cells over-expressing BRAF V600E, while di-theaninate-amido-TAASGVRSMH and thiazole-2-amido-TAASGVRSMH conjugates may be developed for targeting MERTK receptors. These studies provide insight into the molecular interactions of natural product-peptide conjugates and their potential for binding to and targeting MERTK and BRAF V600E receptors in developing new therapeutics for targeting cancer.
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Simulação de Dinâmica Molecular , Proteínas Proto-Oncogênicas B-raf , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , c-Mer Tirosina Quinase/metabolismo , Simulação de Acoplamento Molecular , Linhagem Celular Tumoral , Peptídeos , Tiazóis , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , MutaçãoRESUMO
Recent studies have shown that Ephrin receptors may be upregulated in several types of cancers including breast, ovarian and endometrial cancers, making them a target for drug design. In this work, we have utilized a target-hopping approach to design new natural product-peptide conjugates and examined their interactions with the kinase-binding domain of EphB4 and EphB2 receptors. The peptide sequences were generated through point mutations of the known EphB4 antagonist peptide TNYLFSPNGPIA. Their anticancer properties and secondary structures were analyzed computationally. Conjugates of most optimum of peptides were then designed by binding the N-terminal of the peptides with the free carboxyl group of the polyphenols sinapate, gallate and coumarate, which are known for their inherent anticancer properties. To investigate if these conjugates have a potential to bind to the kinase domain, we carried out docking studies and MMGBSA free energy calculations of the trajectories based on the molecular dynamics simulations, with both the apo and the ATP bound kinase domains of both receptors. In most cases binding interactions occurred within the catalytic loop region, while in some cases the conjugates were found to spread out across the N-lobe and the DFG motif region. The conjugates were further tested for prediction of pharmacokinetic properties using ADME studies. Our results indicated that the conjugates were lipophilic and MDCK permeable with no CYP interactions. These findings provide an insight into the molecular interactions of these peptides and conjugates with the kinase domain of the EphB4 and EphB2 receptor. As a proof of concept, we synthesized and carried out SPR analysis with two of the conjugates (gallate-TNYLFSPNGPIA and sinapate-TNYLFSPNGPIA). Results indicated that the conjugates showed higher binding with the EphB4 receptor and minimal binding to EphB2 receptor. Sinapate-TNYLFSPNGPIA showed inhibitory activity against EphB4. These studies reveal that some of the conjugates may be developed for further investigation into in vitro and in vivo studies and potential development as therapeutics.
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In this work, for the first time, we designed derivatives of beta-D-glucosyloxy-3-hydroxy-trans-stiblene-2-carboxylic acid (GHS), by conjugating GHS with tumor targeting peptides RPARPAR and GGKRPAR to target over-expressed receptors in tumor cells. The sequences RPARPAR and GGKRPAR are known to target the neuropilin1 (NRP1) receptor due to the C-terminal Arg domain; however, their effectiveness has never been examined with other commonly over-expressed receptors in tumor cells, particularly of chronic lymphocytic leukemia that include integrin α1ß1 and CD22. By conjugating these peptides with GHS, which is known for its inherent anti-cancer properties, the goal is to further enhance tumor cell targeting by developing compounds that can target multiple receptors. The physicochemical properties of the conjugates and individual peptides were analyzed using Turbomole and COSMOthermX20 in order to determine their hydrogen bond accepting and donating capabilities. The web server POCASA was used in order to determine the surface cavities and binding pockets of the three receptors. To explore the binding affinities, we conducted molecular docking studies with the peptides and the conjugates with each of the receptors. After molecular docking, the complexes were analyzed using Protein-Ligand Interaction Profiler to determine the types of interactions involved. Molecular dynamics simulation studies were conducted to explore the stability of the receptor-ligand complexes. Our results indicated that in most cases the conjugates showed higher binding and stability with the receptors. Additionally, highly stable complexes of conjugates were obtained with CD22, NRP1 and in most cases with the integrin α1ß1 receptor as well. The binding energies were calculated for each of the receptor ligand complexes through trajectory analysis using MMGBSA studies. SwissADME studies revealed that the compounds showed low GI absorption and were not found to be CYP inhibitors and had bioavailability score that would allow them to be considered as potential drug candidates. Overall, our results for the first time show that the designed conjugates can target multiple over-expressed receptors in tumor cells and may be potentially developed as future therapeutics for targeting tumor cells.
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Simulação de Dinâmica Molecular , Estilbenos , Ácidos Carboxílicos , Integrina alfa1beta1 , Ligantes , Simulação de Acoplamento Molecular , Peptídeos/química , Peptídeos/farmacologia , Ligação ProteicaRESUMO
The inverse problem associated with fitting parameters of an ordinary differential equation (ODE) system to data is nonlinear and multimodal, which is of great challenge to gradient-based optimizers. Markov Chain Monte Carlo (MCMC) techniques provide an alternative approach to solving these problems and can escape local minima by design. APT-MCMC was created to allow users to setup ODE simulations in Python and run as compiled C++ code. It combines affine-invariant ensemble of samplers and parallel tempering MCMC techniques to improve the simulation efficiency. Simulations use Bayesian inference to provide probability distributions of parameters, which enable analysis of multiple minima and parameter correlation. Benchmark tests result in a 20×-60× speedup but 14% increase in memory usage against emcee, a similar MCMC package in Python. Several MCMC hyperparameters were analyzed: number of temperatures, ensemble size, step size, and swap attempt frequency. Heuristic tuning guidelines are provided for setting these hyperparameters.
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OBJECTIVES: Sepsis therapies have proven to be elusive because of the difficulty of translating biologically sound and effective interventions in animal models to humans. A part of this problem originates from the fact that septic patients present at various times after the onset of sepsis, whereas the exact time of infection is controlled in animal models. We sought to determine whether data mining longitudinal physiologic data in a nonhuman primate model of Escherichia coli-induced sepsis could help inform the time of onset of infection. DESIGN: A nearest-neighbor approach was used to back cast the time of onset of infection in animal models of sepsis. Animal data were censored to simulate prospective monitoring at any moment along the septic infection. This was compared against an uncensored database to find the most similar animal in order to estimate the infection onset time. Leave-one-out cross-validation was used for validation. Biomarker selection was performed based on the criteria of estimation accuracy and/or ease of measurement. SETTING: Computational experimental on existing experimental data. SUBJECTS: Retrospective data from 33 septic baboons (Papio ursinus) subjected to Escherichia coli infusion. Validation was performed using 14 pigs that were subjected to surgically induced fecal peritonitis and 22 pigs that were subjected to lipopolysaccharide infusion. MEASUREMENTS AND MAIN RESULTS: Longitudinal physiologic and serum markers, time of death. The presence of uniquely changing biomarkers during septic infection enabled the estimation of infection onset time in the datasets. Various combinations of temporal biomarkers, such as WBC, oxygen content, mean arterial pressure, and heart rate, yielded estimation accuracies of up to 97.8%. The use of temporal vital signs and a single measurement of serum biomarkers yielded highly accurate estimates without the need for invasive measurements. Validation in the pig data revealed similar results despite the heterogeneity of multiple experimental cohorts. This suggests that the method may be effective if sufficiently similar subjects are present in the database. CONCLUSIONS: One nearest-neighbor analysis showed promise in accurately identifying the onset of infection given a database of known infection times and of sufficient breadth. We suggest that this approach is ready for evaluation within the clinical setting using human data.
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Algoritmos , Mineração de Dados , Infecções por Escherichia coli/complicações , Sepse/fisiopatologia , Animais , Pressão Arterial , Biomarcadores/sangue , Biologia Computacional , Modelos Animais de Doenças , Frequência Cardíaca , Contagem de Leucócitos , Oxigênio/sangue , Papio , Reconhecimento Automatizado de Padrão , Estudos Retrospectivos , Sepse/microbiologia , Suínos , Fatores de TempoRESUMO
One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine.
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Células Epiteliais/imunologia , Tolerância Imunológica/imunologia , Timo/crescimento & desenvolvimento , Transplante Homólogo , Aloenxertos/imunologia , Animais , Bioengenharia , Células Epiteliais/citologia , Camundongos , Organoides/imunologia , Medicina Regenerativa , Timo/citologia , Timo/imunologiaRESUMO
Peptide based nanomaterials have been gaining increased prominence due to their ability to form permeable scaffolds that promote growth and regeneration of new tissue. In this work for the first time a short hexapeptide motif VQIVYK, derived from the Tau protein family was conjugated with an organic polyamine linker, putrescine and utilized as a template for developing new materials for cartilage tissue regeneration. Our results showed that the conjugate formed extensive nanofibrous assemblies upon self-assembly under aqueous conditions. We then employed the layer-by-layer (LBL) approach to design the scaffold by first incorporating a short segment of the dentin sialophosphoprotein motif GDASYNSDESK followed by integration with the peptide sequence GSGAGAGSGAGAGSGAGA. This sequence mimics Ala, Gly, Ser repeats seen in the spider silk protein. We then incorporated the polysaccharide alginate which served as a hydrogel. To further enhance binding interactions with chondrocytes, and promote the formation of cartilage in vitro, the bionanocomposites were then attached to the chondrocyte binding peptide sequence HDSQLEALIKFM. The thermal properties as well as biodegradability of the scaffold was examined. To confirm biocompatibility, we examined cell viability, attachment and morphology in the presence of bovine chondrocytes. The cells were found to efficiently adhere to the scaffolds which formed an intricate mesh mimicking the extracellular matrix of cartilage tissue. To evaluate if differentiation occurred in the presence of the scaffolds, we examined in vitro deposition of proteoglycans. Thus, we have developed a new family of nanoscale scaffolds that may be utilized for cartilage tissue regeneration.
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Alginatos/farmacologia , Cartilagem/efeitos dos fármacos , Peptídeos/farmacologia , Regeneração , Alginatos/química , Sequência de Aminoácidos , Animais , Cartilagem/fisiologia , Bovinos , Células Cultivadas , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Microscopia Eletrônica de Varredura , Peptídeos/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
MOTIVATION: Maintenance of the self-renewal state in human embryonic stem cells (hESCs) is the foremost critical step for regenerative therapy applications. The insulin-mediated PI3K/AKT pathway is well appreciated as being the central pathway supporting hESC self-renewal; however, the regulatory interactions in the pathway that maintain cell state are not yet known. Identification of these regulatory pathway components will be critical for designing targeted interventions to facilitate a completely defined platform for hESC propagation and differentiation. Here, we have developed a systems analysis approach to identify regulatory components that control PI3K/AKT pathway in self-renewing hESCs. RESULTS: A detailed mathematical model was adopted to explain the complex regulatory interactions in the PI3K/AKT pathway. We evaluated globally sensitive processes of the pathway in a computationally efficient manner by replacing the detailed model by a surrogate meta-model. Our mathematical analysis, supported by experimental validation, reveals that negative regulators of the molecules IRS1 and PIP3 primarily govern the steady state of the pathway in hESCs. Among the regulators, negative feedback via IRS1 reduces the sensitivity of various reactions associated with direct trunk of the pathway and also constraints the propagation of parameter uncertainty to the levels of post receptor signaling molecules. Furthermore, our results suggest that inhibition of negative feedback can significantly increase p-AKT levels and thereby, better support hESC self-renewal. Our integrated mathematical modeling and experimental workflow demonstrates the significant advantage of computationally efficient meta-model approaches to detect sensitive targets from signaling pathways. AVAILABILITY AND IMPLEMENTATION: FORTRAN codes for the PI3K/AKT pathway and the RS-HDMR implementation are available from the authors upon request.
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Células-Tronco Embrionárias/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Retroalimentação Fisiológica , Humanos , Modelos Biológicos , Fosfatos de Fosfatidilinositol/metabolismo , Análise de SistemasRESUMO
The precise inflammatory role of the cytokine interleukin (IL)-6 and its utility as a biomarker or therapeutic target have been the source of much debate, presumably due to the complex pro- and anti-inflammatory effects of this cytokine. We previously developed a nonlinear ordinary differential equation (ODE) model to explain the dynamics of endotoxin (lipopolysaccharide; LPS)-induced acute inflammation and associated whole-animal damage/dysfunction (a proxy for the health of the organism), along with the inflammatory mediators tumor necrosis factor (TNF)-α, IL-6, IL-10, and nitric oxide (NO). The model was partially calibrated using data from endotoxemic C57Bl/6 mice. Herein, we investigated the sensitivity of the area under the damage curve (AUCD) to the 51 rate parameters of the ODE model for different levels of simulated LPS challenges using a global sensitivity approach called Random Sampling High Dimensional Model Representation (RS-HDMR). We explored sufficient parametric Monte Carlo samples to generate the variance-based Sobol' global sensitivity indices, and found that inflammatory damage was highly sensitive to the parameters affecting the activity of IL-6 during the different stages of acute inflammation. The AUCIL6 showed a bimodal distribution, with the lower peak representing healthy response and the higher peak representing sustained inflammation. Damage was minimal at low AUCIL6, giving rise to a healthy response. In contrast, intermediate levels of AUCIL6 resulted in high damage, and this was due to the insufficiency of damage recovery driven by anti-inflammatory responses from IL-10 and the activation of positive feedback sustained by IL-6. At high AUCIL6, damage recovery was interestingly restored in some population of simulated animals due to the NO-mediated anti-inflammatory responses. These observations suggest that the host's health status during acute inflammation depends in a nonlinear fashion on the magnitude of the inflammatory stimulus, on the host's propensity to produce IL-6, and on NO-mediated downstream responses.
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Interleucina-6/biossíntese , Modelos Teóricos , Doença Aguda , Humanos , Modelos Estatísticos , Método de Monte Carlo , Dinâmica não LinearRESUMO
Robustness is a critical feature of signaling pathways ensuring signal propagation with high fidelity in the event of perturbations. Here we present a detailed quantitative analysis of robustness in insulin mediated PI3K/AKT pathway, a critical signaling pathway maintaining self-renewal in human embryonic stem cells. Using global sensitivity analysis, we identified robustness promoting mechanisms that ensure (1) maintenance of a first order or overshoot dynamics of self-renewal molecule, p-AKT and (2) robust transfer of signals from oscillatory insulin stimulus to p-AKT in the presence of noise. Our results indicate that negative feedback controls the robustness to most perturbations. Faithful transfer of signal from the stimulating ligand to p-AKT occurs even in the presence of noise, albeit with signal attenuation and high frequency cut-off. Negative feedback contributes to signal attenuation, while positive regulators upstream of PIP3 contribute to signal amplification. These results establish precise mechanisms to modulate self-renewal molecules like p-AKT.
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Type 2 diabetes mellitus is a long-lasting endocrine disorder characterized by persistent hyperglycaemia, which is often triggered by an entire or relative inadequacy of insulin production or insulin resistance. As a result of resistance to insulin (IR) and an overall lack of insulin in the body, type 2 diabetes mellitus (T2DM) is a metabolic illness that is characterized by hyperglycaemia. Notably, the occurrence of vascular complications of diabetes and the advancement of IR in T2DM are accompanied by dysbiosis of the gut microbiota. Due to the difficulties in managing the disease and the dangers of multiple accompanying complications, diabetes is a chronic, progressive immune-mediated condition that plays a significant clinical and health burden on patients. The frequency and incidence of diabetes among young people have been rising worldwide. The relationship between the gut microbiota composition and the physio-pathological characteristics of T2DM proposes a novel way to monitor the condition and enhance the effectiveness of therapies. Our knowledge of the microbiota of the gut and how it affects health and illness has changed over the last 20 years. Species of the genus Eubacterium, which make up a significant portion of the core animal gut microbiome, are some of the recently discovered 'generation' of possibly helpful bacteria. In this article, we have focused on pathogenesis and therapeutic approaches towards T2DM, with a special reference to gut bacteria from ancient times to the present day.
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Diabetes Mellitus Tipo 2 , Comportamento Alimentar , Microbioma Gastrointestinal , Diabetes Mellitus Tipo 2/microbiologia , Humanos , Microbioma Gastrointestinal/fisiologia , Animais , Comportamento Alimentar/fisiologia , Disbiose , Resistência à Insulina/fisiologiaRESUMO
A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri. The enzyme exists in a dimer-tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.
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Trifosfato de Adenosina , Ligação Proteica , Ribonucleotídeo Redutases , Ribonucleotídeo Redutases/metabolismo , Ribonucleotídeo Redutases/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Anaerobiose , Nucleotídeos de Desoxiadenina/metabolismo , Domínio Catalítico , Conformação Proteica , Especificidade por Substrato , Multimerização Proteica , Modelos MolecularesRESUMO
Preclinical and clinical studies suggest that lipid-induced hepatic insulin resistance is a primary defect that predisposes to dysfunction in pancreatic islets, implicating a perturbed liver-pancreas axis underlying the comorbidity of T2DM and MASLD. To investigate this hypothesis, we developed a human biomimetic microphysiological system (MPS) coupling our vascularized liver acinus MPS (vLAMPS) with primary islets on a chip (PANIS) enabling MASLD progression and islet dysfunction to be quantitatively assessed. The modular design of this system (vLAMPS-PANIS) allows intra-organ and inter-organ dysregulation to be deconvoluted. When compared to normal fasting (NF) conditions, under early metabolic syndrome (EMS) conditions, the standalone vLAMPS exhibited characteristics of early stage MASLD, while no significant differences were observed in the standalone PANIS. In contrast, with EMS, the coupled vLAMPS-PANIS exhibited a perturbed islet-specific secretome and a significantly dysregulated glucose stimulated insulin secretion (GSIS) response implicating direct signaling from the dysregulated liver acinus to the islets. Correlations between several pairs of a vLAMPS-derived and a PANIS-derived secreted factors were significantly altered under EMS, as compared to NF conditions, mechanistically connecting MASLD and T2DM associated hepatic factors with islet-derived GLP-1 synthesis and regulation. Since vLAMPS-PANIS is compatible with patient-specific iPSCs, this platform represents an important step towards addressing patient heterogeneity, identifying complex disease mechanisms, and advancing precision medicine.
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CONTEXT: Ephrin type A receptor 2 (EphA2) is a well-known drug target for cancer treatment due to its overexpression in numerous types of cancers. Thus, it is crucial to determine the binding interactions of this receptor with both the ligand-binding domain (LBD) and the kinase-binding domain (KBD) through a targeted approach in order to modulate its activity. In this work, natural terpenes with inherent anticancer properties were conjugated with short peptides YSAYP and SWLAY that are known to bind to the LBD of EphA2 receptor. We examined the binding interactions of six terpenes (maslinic acid, levopimaric acid, quinopimaric acid, oleanolic, polyalthic, and hydroxybetulinic acid) conjugated to the above peptides with the ligand-binding domain (LBD) of EphA2 receptor computationally. Additionally, following the "target-hopping approach," we also examined the interactions of the conjugates with the KBD. Our results indicated that most of the conjugates showed higher binding interactions with the EphA2 kinase domain compared to LBD. Furthermore, the binding affinities of the terpenes increased upon conjugating the peptides with the terpenes. In order to further investigate the specificity toward EphA2 kinase domain, we also examined the binding interactions of the terpenes conjugated to VPWXE (x = norleucine), as VPWXE has been shown to bind to other RTKs. Our results indicated that the terpenes conjugated to SWLAY in particular showed high efficacy toward binding to the KBD. We also designed conjugates where in the peptide portion and the terpenes were separated by a butyl (C4) group linker to examine if the binding interactions could be enhanced. Docking studies showed that the conjugates with linkers had enhanced binding with the LBD compared to those without linkers, though binding remained slightly higher without linkers toward the KBD. As a proof of concept, maslinate and oleanolate conjugates of each of the peptides were then tested with F98 tumor cells which are known to overexpress EphA2 receptor. Results indicated that the oleanolate-amido-SWLAY conjugates were efficacious in reducing the cell proliferation of the tumor cells and may be potentially developed and further studied for targeting tumor cells overexpressing the EphA2 receptor. To test if these conjugates could bind to the receptor and potentially function as kinase inhibitors, we conducted SPR analysis and ADP-Glo assay. Our results indicated that OA conjugate with SWLAY showed the highest inhibition. METHODS: Docking studies were carried out using AutoDock Vina, v.1.2.0; Molecular Dynamics and MMGBSA calculations were carried out through Schrodinger Software DESMOND.
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Receptor EphA2 , Receptor EphA2/química , Receptor EphA2/metabolismo , Terpenos/farmacologia , Ligantes , Peptídeos/química , Ligação ProteicaRESUMO
Diabetes is an emerging global epidemic that affects more that 285 million people worldwide. Engineering of endocrine pancreas tissue holds great promise for the future of diabetes therapy. Here we demonstrate the feasibility of re-engineering decellularized organ scaffolds using regenerative cell source. We differentiated human pluripotent stem cells (hPSC) toward pancreatic progenitor (PP) lineage and repopulated decellularized organ scaffolds with these hPSC-PP cells. We observed that hPSCs cultured and differentiated as aggregates are more suitable for organ repopulation than isolated single cell suspension. However, recellularization with hPSC-PP aggregates require a more extensive vascular support, which was found to be superior in decellularized liver over the decellularized pancreas scaffolds. Upon continued culture for nine days with chemical induction in the bioreactor, the seeded hPSC-PP aggregates demonstrated extensive and uniform cellular repopulation and viability throughout the thickness of the liver scaffolds. Furthermore, the decellularized liver scaffolds was supportive of the endocrine cell fate of the engrafted cells. Our novel strategy to engineer endocrine pancreas construct is expected to find potential applications in preclinical testing, drug discovery and diabetes therapy.
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Diabetes Mellitus , Ilhotas Pancreáticas , Células-Tronco Pluripotentes , Humanos , Alicerces Teciduais , Pâncreas , Engenharia Tecidual , Matriz ExtracelularRESUMO
Development of biocomposite scaffolds has gained tremendous attention due to their potential for tissue regeneration. However, most scaffolds often contain animal-derived collagen that may elicit an immunological response, necessitating the development of new biomaterials. Herein, we developed a new collagen-like peptide,(Pro-Ala-His)10 (PAH)10, and explored its ability to be utilized as a functional biomaterial by incorporating it with a newly synthesized peptide-based self-assembled gel. The gel was prepared by conjugating a pectin derivative, galataric acid, with a pro-angiogenic peptide (LHYQDLLQLQY) and further functionalized with a cortistatin-derived peptide, (Phe-Trp-Lys-Thr)4 (FWKT)4, and the bio-ionic liquid choline acetate. The self-assembly of (PAH)10 and its interactions with the galactarate-peptide conjugates were examined using replica exchange molecular dynamics (REMD) simulations. Results revealed the formation of a multi-layered scaffold, with enhanced stability at higher temperatures. We then synthesized the scaffold and examined its physicochemical properties and its ability to integrate with aortic smooth muscle cells. The scaffold was further utilized as a bioink for bioprinting to form three-dimensional cell-scaffold matrices. Furthermore, the formation of actin filaments and elongated cell morphology was observed. These results indicate that the (PAH)10 hybrid scaffold provides a suitable environment for cell adhesion, proliferation and growth, making it a potentially valuable biomaterial for tissue engineering.
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Tissue engineering (TE) aims to repair and regenerate damaged tissue by an assimilation of optimal combination of cells specific to the tissue with an appropriate biomaterial. In this work, a new biomaterial for potential cardiac TE applications was developed by utilizing a combination of in silico studies and in vitro experiments. Molecular dynamics (MD) simulations for the formation of the novel composite prepared from the decellularized leaf components cellulose and pectin along with the VEGF derived peptide (NYLTHRQ) and polypyrrole (PPy) was carried out to assess self-assembly, mechanical properties, and interactions with integrin and NPR-C receptors which are commonly found in cells of cardiac tissue. Results of molecular dynamics simulations indicated the successful formation of stable assemblies. MD simulations also revealed that the scaffold successfully interacted with integrin and NPR-C receptors. As a proof of concept, beet leaves were decellularized (DC) and cross-linked with NYLTHRQ and PPy using layer-by-layer assembly. Decellularization (DC) was confirmed by DNA and protein quantification. Incorporation of the NYLTHRQ peptide and polypyrrole was confirmed by FTIR spectroscopy and SEM imaging. The DC-NYLTHRQ-PPy scaffold was seeded with co-cultured cardiomyocytes and vascular smooth muscle cells. The scaffold promoted cell proliferation and adhesion. Actin and Troponin T immunofluorescence staining showed the presence of these critical cardiomyocyte markers. Thus, for the first time we have developed a decellularized leaf-peptide-PPy composite scaffold by a combination of in silico studies and laboratory analyses that may have potential applications in cardiac TE.Communicated by Ramaswamy H. Sarma.
Assuntos
Polímeros , Engenharia Tecidual , Engenharia Tecidual/métodos , Polímeros/química , Pirróis/química , Simulação de Dinâmica Molecular , Materiais Biocompatíveis , Peptídeos , IntegrinasRESUMO
Peptide nanoassemblies have garnered remarkable importance in the development of novel nanoscale biomaterials for drug delivery into tumor cells. Taking advantage of receptor mediated recognition of two known peptides, angiopep-2 (TFFYGGSRGKRNNFKTEEY) and A-COOP-K (ACGLSGLC10 VAK) that bind to the over-expressed receptors low density lipoprotein (LRP-1) and fatty acid binding protein (FABP3) respectively, we have developed new peptide conjugates by combining the anti-inflammatory, antitumor compound azelaic acid with angiopep-2, which efficiently self-assembled into nanofibers. Those nanofibers were then functionalized with the A-COOP-K sequence and formed supramolecular hierarchical structures that were found to entrap the chemotherapeutic drug doxorubicin efficaciously. Furthermore, the nanoassemblies were found to release the drug in a dose-dependent manner and showed a stepwise increase over a period of 2 weeks under acidic conditions. Two cell lines (U-87-MG and U-138-MG) were utilized as models for glioblastoma cells grown in the presence of serum and under serum-free conditions to mimic the growth conditions of natural tumors. The drug entrapped assemblies were found to inhibit the cell proliferation of both U-87 and U-138MG glioblastoma cells. Three dimensional spheroids of different sizes were grown to mimic the tumors and evaluate the efficacy of drug release and internalization. Our results indicated that the nanoassemblies were found to have higher internalization of DOX and were well-spread throughout the spheroids grown, particularly under serum-free conditions. The nanoassemblies also displayed blood-brain barrier penetration when tested with a multicellular in vitro model. Such self-assembled nanostructures with targeting ability may provide a suitable platform for the development of new peptide-based biomaterials that can provide more insights about the mechanistic approach for drug delivery for not only 2D cell cultures but also 3D tumoroids that mimic the tumor microenvironments.