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Fibroblast growth factor 21 (FGF21) is pivotal in regulating energy metabolism, highlighting substantial therapeutic potential for non-alcoholic steatohepatitis (NASH). Previously, we reported a long-acting FGF21 fusion protein, PsTag-FGF21, which was prepared by genetically fusing human FGF21 with a 648-residue polypeptide (PsTag). While this fusion protein demonstrated therapeutic efficacy against NASH, our final product analysis revealed the presence of fixed impurities resistant to effective removal, indicating potential degradation of PsTag-FGF21. Here, we enriched and analyzed the impurities, confirming our hypothesis regarding the C-terminal degradation of PsTag-FGF21. We now describe a new variant developed to eliminate the C-terminal degradation. By introducing one mutation located at the C-terminal of PsTag-FGF21(V169L), we demonstrated that the new molecule, PsTag-FGF21(V169L), exhibits many improved attributes. Compared with PsTag-FGF21, PsTag-FGF21(V169L) displayed elevated bioactivity and stability, along with a twofold enhanced binding affinity to the coreceptor ß-Klotho. In vivo, the circulating half-life of PsTag-FGF21(V169L) was further enhanced compared with that of PsTag-FGF21. In NASH mice, PsTag-FGF21(V169L) demonstrated efficacy with sustained improvements in multiple metabolic parameters. Besides, PsTag-FGF21(V169L) demonstrated the ability to alleviate NASH by decreasing hepatocyte apoptosis. The superior biophysical, pharmacokinetic, and pharmacodynamic properties, along with the positive metabolic effects, imply that further clinical development of PsTag-FGF21(V169L) as a metabolic therapy for NASH patients may be warranted.
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Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease globally. Non-alcoholic steatohepatitis (NASH) represents an extremely progressive form of NAFLD, which, without timely intervention, may progress to cirrhosis or hepatocellular carcinoma. Presently, a definitive comprehension of the pathogenesis of NAFLD/NASH eludes us, and pharmacological interventions targeting NASH specifically remain constrained. The aetiology of NAFLD encompasses a myriad of external factors including environmental influences, dietary habits and gender disparities. More significantly, inter-organ and cellular interactions within the human body play a role in the development or regression of the disease. In this review, we categorize the influences affecting NAFLD both intra- and extrahepatically, elaborating meticulously on the mechanisms governing the onset and progression of NAFLD/NASH. This exploration delves into progress in aetiology and promising therapeutic targets. As a metabolic disorder, the development of NAFLD involves complexities related to nutrient metabolism, liver-gut axis interactions and insulin resistance, among other regulatory functions of extraneous organs. It further encompasses intra-hepatic interactions among hepatic cells, Kupffer cells (KCs) and hepatic stellate cells (HSCs). A comprehensive understanding of the pathogenesis of NAFLD/NASH from a macroscopic standpoint is instrumental in the formulation of future therapies for NASH.
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Células Estrelladas Hepáticas , Macrófagos del Hígado , Hígado , Enfermedad del Hígado Graso no Alcohólico , Humanos , Enfermedad del Hígado Graso no Alcohólico/patología , Macrófagos del Hígado/metabolismo , Hígado/patología , Hígado/metabolismo , Células Estrelladas Hepáticas/metabolismo , Resistencia a la Insulina , Progresión de la Enfermedad , AnimalesRESUMEN
BACKGROUND AND PURPOSE: Because of the absence of effective therapies for metabolic dysfunction-associated steatohepatitis (MASH), there is a rising interest in fibroblast growth factor 21 (FGF21) analogues due to their potential anti-fibrotic activities in MASH treatment. PsTag-FGF21, a long-acting FGF21 analogue, has demonstrated promising therapeutic effects in several MASH mouse models. However, its efficacy and mechanism against MASH-related fibrosis remain less well defined, compared with the specific mechanisms through which FGF21 improves glucose and lipid metabolism. EXPERIMENTAL APPROACH: The effectiveness of PsTag-FGF21 was evaluated in two MASH-fibrosis models. Co-culture systems involving macrophages and hepatic stellate cells (HSCs) were employed for further assessment. Hepatic macrophages were selectively depleted by administering liposome-encapsulated clodronate via tail vein injections. RNA sequencing and cytokine profiling were conducted to identify key factors involved in macrophage-HSC crosstalk. KEY RESULTS: We first demonstrated the significant attenuation of hepatic fibrosis by PsTag-FGF21 in two MASH-fibrosis models. Furthermore, we highlighted the crucial role of macrophage phenotypic switch in PsTag-FGF21-induced HSC deactivation. FGF21 was demonstrated to regulate macrophages in a PsTag-FGF21-like manner. NR4A1, a nuclear factor which is notably down-regulated in human livers with MASH, was identified as a mediator responsible for PsTag-FGF21-induced phenotypic switch. Transcriptional control over insulin-like growth factor 1, a crucial factor in macrophage-HSC crosstalk, was exerted by the intrinsically disordered region domain of NR4A1. CONCLUSION AND IMPLICATIONS: Our results have elucidated the previously unclear mechanisms through which PsTag-FGF21 treats MASH-related fibrosis and identified NR4A1 as a potential therapeutic target for fibrosis.
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Factores de Crecimiento de Fibroblastos , Macrófagos , Ratones Endogámicos C57BL , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Animales , Ratones , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Humanos , Fenotipo , Hígado Graso/tratamiento farmacológico , Hígado Graso/metabolismo , Células Cultivadas , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismoRESUMEN
Altered glycosylation profiles have been correlated with potential drug targets in various diseases, including Alzheimer's disease (AD). In this area, the linkage between bisecting N-acetylglucosamine (GlcNAc), a product of N-acetylglucosaminyltransferase III (GnT-III), and AD has been recognized, however, our understanding of the cause and the causative role of this aberrant glycosylation in AD are far from completion. Moreover, the effects and mechanisms of glycosylation-targeting interventions on memory and cognition, and novel targeting strategies are worth further study. Here, we showed the characteristic amyloid pathology-induced and age-related changes of GnT-III, and identified transcription factor 7-like 2 as the key transcription factor responsible for the abnormal expression of GnT-III in AD. Upregulation of GnT-III aggravated cognitive dysfunction and Alzheimer-like pathologies. In contrast, loss of GnT-III could improve cognition and alleviate pathologies. Furthermore, we found that an increase in bisecting GlcNAc modified ICAM-1 resulted in impairment of microglial responses, and genetic inactivation of GnT-III protected against AD mechanistically by blocking the aberrant glycosylation of ICAM-1 and subsequently modulating microglial responses, including microglial motility, phagocytosis ability, homeostatic/reactive state and neuroinflammation. Moreover, by target-based screening of GnT-III inhibitors from FDA-approved drug library, we identified two compounds, regorafenib and dihydroergocristine mesylate, showing pharmacological potential leading to modulation of aberrant glycosylation and microglial responses, and rescue of memory and cognition deficits.
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Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/metabolismo , Glicosilación , Molécula 1 de Adhesión Intercelular/metabolismo , Microglía/metabolismo , CogniciónRESUMEN
Hepatitis B virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC). An interesting question is how different are the molecular and phenotypic profiles between HBV-infected (HBV+) and non-HBV-infected (HBV-) HCCs? Based on the publicly available multi-omics data for HCC, including bulk and single-cell data, and the data we collected and sequenced, we performed a comprehensive comparison of molecular and phenotypic features between HBV+ and HBV- HCCs. Our analysis showed that compared to HBV- HCCs, HBV+ HCCs had significantly better clinical outcomes, higher degree of genomic instability, higher enrichment of DNA repair and immune-related pathways, lower enrichment of stromal and oncogenic signaling pathways, and better response to immunotherapy. Furthermore, in vitro experiments confirmed that HBV+ HCCs had higher immunity, PD-L1 expression and activation of DNA damage response pathways. This study may provide insights into the profiles of HBV+ and HBV- HCCs, and guide rational therapeutic interventions for HCC patients.
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Carcinoma Hepatocelular , Virus de la Hepatitis B , Neoplasias Hepáticas , Carcinoma Hepatocelular/virología , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/virología , Neoplasias Hepáticas/genética , Humanos , Virus de la Hepatitis B/genética , Fenotipo , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Hepatitis B/virología , Hepatitis B/complicaciones , Hepatitis B/genética , Inestabilidad Genómica , Reparación del ADN , MultiómicaRESUMEN
Despite the clinical breakthrough made by immune checkpoint blockades (ICB) in cancer immunotherapy, immunosuppressed tumor microenvironment (TME) remains a major impediment in the efficacy of ICB immunotherapy. In this study, we constructed a Nitrated T cell epitope (NitraTh) linked vaccine targeting CD47, namely CD47-NitraTh. CD47-NitraTh could repress the progression of tumor by inducing tumor-specific immune response. Furthermore, combination vaccination with CD47-NitraTh and PDL1-NitraTh could reconstruct tumor associated macrophage, enhance macrophage-mediated phagocytosis for tumor cells, and promote the activation of tumor infiltrating T cells. Notably, by activating chemokine signaling pathway, NitraTh based vaccines reversed immunosuppressed TME, resulting in improved therapeutic outcome for tumor. With the advantage of reversing immunosuppressed TME, NitraTh based vaccine seems an optimal immunotherapy strategy for patients who are not sensitive to antibody based ICB.
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Vacunas contra el Cáncer , Neoplasias , Humanos , Antígeno CD47 , Epítopos de Linfocito T , Inmunoterapia/métodos , Nitratos , Fagocitosis , Microambiente Tumoral , Vacunas contra el Cáncer/inmunologíaRESUMEN
INTRODUCTION: Lacosamide is licensed for the treatment of focal seizures in both adults and children, however there is little information available on its adverse reactions. Using the FDA Adverse Event Reporting System (FAERS), we seek to assess adverse occurrences that may be related to Lacosamide. METHODS: On the basis of the FAERS database from the fourth quarter of 2008 to the second quarter of 2022, disproportionality analysis was carried out using the reporting odds ratio (ROR) method, the United Kingdom Medicines and Healthcare Products Regulatory Agency omnbius standard (MHRA) method, and the bayesian confidence propagation neural network (BCPNN) method. We extracted valuable positive signals for designated medical event (DME) screening, focused on the evaluation and comparison of safety signals appearing in DME with system organ classification (SOC) analysis. RESULTS: A total of 10,226 adverse reaction reports with Lacosamide as the primary suspect drug were obtained, with 30,960 reported cases, detecting 232 valuable positive signals, involving a total of 20 SOCs, of which the most frequently reported SOCs were nervous system disorders (6537 cases, 55.21%), psychiatric disorders (1530 cases, 12.92%), injury poisoning and procedural complications (1059 cases, 8.94%). According to 232 valuable positive signals with DME screening results, two signals of stevens-johnson syndrome and ventricular fibrillation were consistent with PT signals on the DME list, with the two SOCs focusing on skin and subcutaneous tissue disorders and cardiac disorders, respectively. CONCLUSIONS: Our research demonstrates that the clinical use of Lacosamide should be noticed and avoided in relation to ADRs since it raises the risk of cardiac arrest, ventricular fibrillation, stevens-johnson syndrome, and rhabdomyolysis.
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Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Síndrome de Stevens-Johnson , Adulto , Niño , Humanos , Estados Unidos/epidemiología , Farmacovigilancia , Lacosamida/efectos adversos , Teorema de Bayes , Fibrilación Ventricular , Sistemas de Registro de Reacción Adversa a Medicamentos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/epidemiología , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiologíaRESUMEN
BACKGROUND: There is a serious shortage of clinical pharmacists in Xinjiang, China. A six-month to one-year on-the-job training programme can rapidly transition nonclinical pharmacists into clinical pharmacists to resolve this issue. However, not all nonclinical pharmacists are willing to become clinical pharmacists, and many factors may influence their willingness. This study aims to assess the transformation intention of nonclinical hospital pharmacists and the contributing elements to make recommendations to accelerate the transformation of hospital pharmacists to clinical pharmacists. METHODS: Cross-sectional survey was conducted in secondary and tertiary hospitals in Xinjiang. Taking 14 prefectures in Xinjiang as a cluster, 34 hospitals were randomly selected. By snowball sampling, the heads of pharmaceutical departments introduced non-clinical pharmacists to participate in an anonymous questionnaire survey, which included 41 questions about basic demographic information, cognition and attitudes towards pharmaceutical care, potential factors and willingness to transform, and it took an average of 10 min to complete. Using multifactor logistic regression, the contributing elements of transformation intention were analysed. RESULTS: The survey was conducted from May to October 2022. 338 valid responses were obtained, with a response rate of 91.85% and a willingness to transform rate of 81.67%. There were significant differences in the willingness to transform among nonclinical pharmacists of different ages (P < 0.05), marital statuses (P < 0.05), years of employment (P < 0.05), and technical titles (P < 0.05). There were significant differences between the two groups in the following four aspects: whether the setting of human resources in the pharmaceutical department was reasonable (P < 0.05), the educational level of clinical pharmacists (P < 0.05), the higher salary level of clinical pharmacists (P < 0.05), and whether they had experience in pharmaceutical care (P < 0.05). There was a significant difference in the total score of the pharmaceutical care attitude scale (P < 0.05) between the willing and unwilling groups. The results of multivariate logistic regression analysis revealed that the experience of providing pharmaceutical care (OR = 4.601, 95% CI: 1.13-18.69, P < 0.05) and attitude towards pharmaceutical care (OR = 3.302, 95% CI: 1.19-9.19, P < 0.05) had a statistically significant influence on the transformation intention of nonclinical pharmacists. CONCLUSIONS: One-fifth of nonclinical pharmacists were unwilling to transition to clinical pharmacists. The attitude towards and experience of pharmaceutical care affected pharmacists' transformation intention, so the suggestion is proposed to promote the transformation of nonclinical pharmacists into clinical pharmacists.
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Actitud del Personal de Salud , Farmacéuticos , Humanos , Estudios Transversales , Encuestas y Cuestionarios , China , Preparaciones FarmacéuticasRESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by selective loss of dopaminergic neurons. We previously found that inhibition of von Hippel-Lindau (VHL) can alleviate dopaminergic neuron degeneration in PD models via regulation of mitochondrial homeostasis, however, the disease-related alterations of VHL and the regulatory mechanisms of VHL level in PD need to be further investigated. In this study, we found that the levels of VHL were markedly increased in multiple cell models of PD and identified microRNA-143-3p (miR-143-3p) as a promising candidate for regulating VHL expression involved in PD. miR-143-3p directly bound to the 3'untranslated region of human VHL mRNA and inhibited its translation, and exerted neuroprotective effects by improving cell viability, apoptosis and tyrosine hydroxylase abnormality. Furthermore, we demonstrated that miR-143-3p exerted neuroprotection by attenuating mitochondrial abnormality via AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) axis, and AMPK inhibitor abolished the beneficial effects of miR-143-3p on the cell model of PD. Therefore, we identify the dysregulated VHL and miR-143-3p in PD, and propose the therapeutic potential of miR-143-3p to alleviate PD by improving mitochondrial homeostasis via AMPK/PGC-1α axis.
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MicroARNs , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Mitocondrias/metabolismo , MicroARNs/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
Neoantigen vaccines constitute an emerging and promising cancer immunotherapy. However, not all neoantigens have anti-tumor activity, as poor CD4+ epitope recognition can lead to the lack of greatly limit the persistence of the CD8+ T cell response. Therefore, we designed a self-assembled nanoplatform hereinafter referred to as DNA-coupled nitrated T helper cell epitope nanoparticle (DCNP) based on DNA origami containing a nitrated CD4 + T cell epitope, which can facilitate the effective activation of neoantigen-specific CD8+ T cells. Moreover, we embedded the cytidine-phosphate-guanosine oligonucleotide (CpG ODN) motif sequence in the DNA skeleton to function as a built-in adjuvant to activate Toll-like receptor 9. DCNP can markedly improve adjuvant and neoantigen co-delivery to lymphoid organs and promote neoantigen presentation on dendritic cells. Moreover, DCNP induced robust, and long-lived neoantigen-specific CD8+ T cell responses that significantly delayed tumor growth. Further, these effects were largely dependent on the nitrated T cell epitope. Collectively, our findings indicate that DCNP is a promising platform that could improve the development of personalized therapeutic neoantigen vaccines for cancer immunotherapy.
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Vacunas contra el Cáncer , Nanopartículas , Neoplasias , Humanos , Epítopos de Linfocito T , Nitratos , Antígenos de Neoplasias , Neoplasias/tratamiento farmacológico , Linfocitos T Colaboradores-Inductores , Adyuvantes Inmunológicos , ADN , InmunoterapiaRESUMEN
BACKGROUND AND PURPOSE: Repeated amino acid sequences in proteins are widely found, and the glycine-serine-alanine repeat is an element with a general propensity to form ß-sheet aggregates as found in key pathological factors, in several neurodegenerative diseases. Such properties of this repeat may guide development of disease-modifying therapies for neurodegenerative disease. However, details of its role and underlying mechanism(s) remain largely unknown. EXPERIMENTAL APPROACH: Actions of specific glycine-serine-alanine repeat peptides (SNPs), especially SNP-9, on Alzheimer's disease (AD)-like abnormalities were evaluated in transgenic mice and Caenorhabditis elegans, and in rat and cell models. Entry of SNPs into the brain, SNP activity in neuronal cells and peptide entry into cells were analysed in vivo and in vitro. Cell-free systems and the yeast two-hybrid system were also used to explore possible targets of SNP-9, and interactions of potential targets with SNP-9 were confirmed in cell-based systems. KEY RESULTS: We first identified SNP-9 as a potent neuroprotective peptide with the activity to decrease oligomeric amyloid ß (Aß) via co-assembling with the toxic Aß oligomer to form hetero-oligomers. Also, calcyclin-binding protein was found to act as a SNP-9-binding protein, by screening of a human brain cDNA library. Such binding showed that SNP-9 could regulate the abnormal hyperphosphorylation of tau via calcyclin-binding protein. CONCLUSION AND IMPLICATIONS: Our study provides a foundation for development of SNPs, especially SNP-9, as potential therapeutic interventions for AD. We propose SNP-9 as a potential therapeutic agent for the treatment of AD.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Ratones , Ratas , Animales , Humanos , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Serina , Proteína A6 de Unión a Calcio de la Familia S100 , Ratones Transgénicos , Caenorhabditis elegans/metabolismoRESUMEN
Dopaminergic neuron degeneration is a hallmark of Parkinson's disease (PD). We previously reported that the inactivation of von HippelâLindau (VHL) alleviated dopaminergic neuron degeneration in a C. elegans model. In this study, we investigated the specific effects of VHL loss and the underlying mechanisms in mammalian PD models. For in vivo genetic inhibition of VHL, AAV-Vhl-shRNA was injected into mouse lateral ventricles. Thirty days later, the mice received MPTP for 5 days to induce PD. Behavioral experiments were conducted on D1, D3, D7, D14 and D21 after the last injection, and the mice were sacrificed on D22. We showed that knockdown of VHL in mice significantly alleviated PD-like syndromes detected in behavioral and biochemical assays. Inhibiting VHL exerted similar protective effects in MPP+-treated differentiated SH-SY5Y cells and the MPP+-induced C. elegans PD model. We further demonstrated that VHL loss-induced protection against experimental parkinsonism was independent of hypoxia-inducible factor and identified the Dishevelled-2 (DVL-2)/ß-catenin axis as the target of VHL, which was evolutionarily conserved in both C. elegans and mammals. Inhibiting the function of VHL promoted the stability of ß-catenin by reducing the ubiquitination and degradation of DVL-2. Thus, in vivo overexpression of DVL-2, mimicking VHL inactivation, protected against PD. We designed a competing peptide, Tat-DDF-2, to inhibit the interaction between VHL and DVL-2, which exhibited pharmacological potential for protection against PD in vitro and in vivo. We propose the therapeutic potential of targeting the interaction between VHL and DVL-2, which may represent a strategy to alleviate neurodegeneration associated with PD.
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Proteínas Dishevelled , Enfermedad de Parkinson , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau , Animales , Humanos , Ratones , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , beta Catenina/metabolismo , Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Proteínas Dishevelled/efectos de los fármacos , Proteínas Dishevelled/metabolismo , Dopamina/farmacología , Neuronas Dopaminérgicas/metabolismo , Mamíferos , Ratones Endogámicos C57BL , Neuroblastoma/metabolismo , Enfermedad de Parkinson/metabolismo , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/metabolismo , Ubiquitinación/efectos de los fármacos , Ubiquitinación/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/antagonistas & inhibidores , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Multidrug resistance (MDR) to chemotherapeutic drugs and targeted drug delivery are recurring issues in clinical cancer treatment. Here, a multifunctional fusion protein-DNA conjugate was designed as a co-delivery vehicle for anticancer peptides and chemotherapeutic drugs to combat both drug-resistant and drug-sensitive tumor cells. The fusion protein was constructed by fusing a PsTag polypeptide, a matrix metalloproteinase 2 (MMP2)-degradable domain, and the mitochondria-targeted pro-apoptotic peptide KLAKLAKKLAKLAK. Doxorubicin was efficiently loaded into the fusion protein pre-conjugated dendrimer-like DNA nanostructure. With the incorporation of enhanced stability, tumor targeting, and controlled-release elements, the tailored nanostructure can selectively enter tumor cells and synergistically exert antitumor activity with no significant adverse effects. Thus, these protein-conjugated DNA nanocarriers could be a potential co-delivery system for protein/peptide and chemotherapeutic drugs delivery in synergistic cancer therapy.
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Antineoplásicos , Sistemas de Liberación de Medicamentos , Neoplasias , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , ADN , Doxorrubicina , Resistencia a Antineoplásicos , Humanos , Metaloproteinasa 2 de la Matriz , Nanopartículas , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Péptidos/químicaRESUMEN
BACKGROUND AND PURPOSE: Multidrug resistance (MDR) is a major obstacle to the successful treatment of cancer. Ample evidence shows that ATP-binding cassette (ABC) transporters and high-energy states in cells are linked to cancer drug resistance. Our previous work reported an engineered therapeutic protein named PAK, which selectively inhibited tumour progression by targeting mitochondria. EXPERIMENTAL APPROACH: We studied the effects of PAK on reversing drug resistance in MDR phenotypic cells and xenograft mouse models. Effects of PAK on the process of mitochondrial energy production, ABC transporter expression, and drugs enrichment were investigated in cancer cells. RNA-seq and co-immunoprecipitation were employed to analyse the mechanism of PAK on the redistribution of ABC transporters. KEY RESULTS: PAK promoted the enrichment of drugs in MDR cancer cells, thus enhancing the sensitivity of cancer cells to chemotherapy. Furthermore, PAK was colocalized in the mitochondria and initiated mitochondrial injury by selectively inhibiting the mitochondrial complex V. Also, ABCB1 and ABCC1 were redistributed from the plasma membrane to the cytoplasm through the disruption of lipid rafts, which was attributed to the low energy state and decreased cholesterol levels. CONCLUSIONS AND IMPLICATIONS: Our results revealed a previously unrecognized pattern of reversal of drug resistance and have suggested mitochondria as a clinically relevant target for the treatment of MDR malignant tumours.
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Antineoplásicos , Neoplasias , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Ratones , Mitocondrias/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
Dual activation of the glucagon-like peptide 1 (GLP-1) receptor and the glucose-dependent insulinotropic polypeptide (GIP) receptor has potential as a novel strategy for treatment of diabesity. Here, we created a hybrid peptide which we named 19W, and show that it is more stable in presence of murine plasma than exendin-4 is. In vitro studies were performed to reveal that 19W could stimulate insulin secretion from INS-1 cells in a dose-dependent manner, just like the native peptide GIP and exendin-4 do. 19W effectively evoked dose-dependent cAMP production in cells targeting both GLP-1R and GIPR. In healthy C57BL/6J mice, the single administration of 19W significantly improved glucose tolerance. When administered in combination with sodium deoxycholate (SDC), its oral hypoglycemic activity was enhanced. Pharmacokinetics studies in Wistar rats revealed that 19W was absorbed following oral uptake, while SDC increased its bioavailability. A long-term (28 days) exposure study of twice-daily oral administration to high fat-fed (HFF) mice showed that 19W significantly reduced animal food intake, body weight, fasting blood glucose, total serum cholesterol (T-CHO), non-esterified free fatty acids (NEFA), and low-density lipoprotein cholesterol (LDL-C) levels. It also significantly improved glucose tolerance and the pancreatic ß/α cell ratio, and decreased the area of liver fibrosis. These results clearly demonstrate the beneficial action of this novel oral GLP-1/GIP dual receptor agonist to reduce adiposity and hyperglycemia in diabetic mice and to ameliorate liver fibrosis associated with obesity. This dual-acting peptide can be considered a good candidate for novel oral therapy to treat obesity and diabetes.
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Péptido 1 Similar al Glucagón/metabolismo , Hipoglucemiantes/farmacología , Secreción de Insulina/efectos de los fármacos , Obesidad , Receptores de la Hormona Gastrointestinal/metabolismo , Animales , AMP Cíclico/biosíntesis , Diabetes Mellitus Experimental , Ingestión de Alimentos/efectos de los fármacos , Exenatida/farmacología , Polipéptido Inhibidor Gástrico/metabolismo , Incretinas/farmacología , Insulina/metabolismo , Enfermedades Metabólicas/tratamiento farmacológico , Enfermedades Metabólicas/metabolismo , Ratones , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , RatasRESUMEN
The activity and function of proteins can be improved by incorporation of non-canonical amino acids (ncAAs). To avoid the tedious synthesis of a large number of chiral phenylalanine derivatives, we synthesized the corresponding phenylpyruvic acid precursors. Escherichia coli strain DH10B and strain C321.ΔA.expΔPBAD were selected as hosts for phenylpyruvic acid bioconversion and genetic code expansion using the MmPylRS/pyltRNACUA system. The concentrations of keto acids, PLP and amino donors were optimized in the process. Eight keto acids that can be biotransformed and their coupled genetic code expansions were identified. Finally, the genetic encoded ncAAs were tested for incorporation into fluorescent proteins with keto acids.
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Código Genético , Cetoácidos/metabolismo , Fenilalanina/genética , Escherichia coli/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Cetoácidos/química , Fenilalanina/química , Fosfato de Piridoxal/metabolismo , Especificidad por SustratoRESUMEN
Despite the well-recognized clinical success of therapeutic proteins, especially antibodies, their inability to penetrate the cell membrane restricts them to secretory extracellular or membrane-associated targets. Developing a direct cytosolic protein delivery system would offer unique opportunities for intracellular target-related therapeutic proteins. Here, we generated a supercharged polypeptide (SCP) with high cellular uptake efficiency, endosomal escape ability, and good biosafety and developed an SCP with an unnatural amino acid containing the phenylboronic acid (PBA) group, called PBA-SCP. PBA-SCP is capable of potently delivering proteins with various isoelectric points and molecular sizes into the cytosol of living cells without affecting their bioactivities. Importantly, cytosolically delivered antibodies remain functional and are capable of targeting, labeling, and manipulating diverse intracellular antigens. This study demonstrates an efficient and versatile intracellular protein delivery platform, especially for antibodies, and provides new possibilities for expanding protein-based therapeutics to intracellular "undruggable" targets.
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Péptidos , Proteínas , Transporte Biológico , Citosol/metabolismo , Endosomas/metabolismo , Péptidos/metabolismoRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disease, the main pathological features include deposition of neurofibrillary tangles composed of the abnormally hyperphosphorylated tau protein and plaques deposition composed of ß-amyloid (Aß) peptide. MicroRNAs and aberrant glycosylation both play key roles in a variety of diseases, especially AD. Our previous study showed that N-acetylglucosaminyltransferase III (GnT-III) was expressed strongly in AD model mice. GnT-III is a glycosyltransferase responsible for synthesizing a bisecting N-acetylglucosamine residue. Here, we report the potential therapeutic effects of microRNA-23b (miR-23b) against AD by targeting GnT-III. In this study, the role of miR-23b in GnT-III-mediated amelioration of AD-related symptoms and pathologies, and mechanisms were investigated. We used Aß1-42-induced mouse and PC12 cell models to evaluate the effects of miR-23b on cognitive impairment, neurotoxicity, tau, and amyloid pathology. Bioinformatics analysis showed that GnT-III may be targeted by miR-23b, and it was verified by dual-luciferase reporter gene assays. Furthermore, a mechanistic study showed that activation of the Akt/GSK-3ß signaling pathway can contribute to tau-lesion inhibition by miR-23b, and miR-23b can also restrain oxidative stress by altering Aß-precursor protein processing. Taken together, we conclude that overexpression of miR-23b can interrupt the pathogenesis of AD.
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Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/toxicidad , Encéfalo/metabolismo , MicroARNs/genética , N-Acetilglucosaminiltransferasas/metabolismo , Fragmentos de Péptidos/toxicidad , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Ratones , MicroARNs/metabolismo , Prueba del Laberinto Acuático de Morris , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Estrés Oxidativo/genética , Células PC12 , Placa Amiloide/metabolismo , Placa Amiloide/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Proteínas tau/metabolismoRESUMEN
Receptor endocytic trafficking entails targeting receptors and ligands to endocytic sites, followed by internalization and sorting to recycling or degradative compartments. Thus, membrane receptor-mediated signalling pathways not only contribute to the efficacy of the drugs but also play a crucial role in the metabolic elimination of peptide drugs. Glucagon-like peptide-1 (GLP-1) receptor is the crucial target for type 2 diabetes mellitus. We mainly focused on the characteristics, early evaluation of GLP-1 receptor endocytosis and effects of optimization for endocytosis on druggability. The GLP-1 receptor endocytosis characteristics of agonists were analysed by a multifunction microplate reader, flow cytometer and confocal microscope. The intracellular cyclic adenosine monophosphate (cAMP) activation of agonists was analysed based on a reporter gene assay, and intracellular ß-arrestin recruitment detection was detected based on a Tango assay. We established quantitative evaluation methods of endocytosis based on fluorescently labelled agonist and receptor trafficking and used them to screen agonists with less endocytosis. Sprague-Dawley rats were used for pharmacokinetic analyses, and the hypoglycaemic activity was evaluated by intraperitoneal glucose tolerance tests (IPGTT). Our results showed that GLP-1 receptor-mediated endocytosis, as a manner of elimination, was clathrin-dependent. More importantly, we found that agonists biased towards the G protein pathway were less endocytosed by GLP-1 receptor. We screened an analogue of Exendin-4 M4, which was biased toward the G protein pathway with less endocytosis by the GLP-1 receptor. M4, which shows prolonged hypoglycaemic activities and a long half-life, can be used as a lead compound for type 2 diabetes mellitus treatment.
Asunto(s)
Hipoglucemiantes , Animales , Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón , RatasRESUMEN
Immunological adjuvants are an important part of tumor vaccines and are critical for stimulating anti-tumor immune responses. However, the clinical needs of strong adjuvants have not been met. In this work, we found that the purified acidic polysaccharide from Sarcandra glabra, named p-SGP, is an ideal adjuvant for tumor vaccines. Cancer vaccines could induce stronger humoral and cellular immune responses when they are adjuvanted with p-SGP. Compared with CpG, a well-studied adjuvant, p-SGP significantly augmented the anti-tumor immunity of various cancer vaccines, which is leading to noticeable inhibition of tumor growth and metastasis in tumor-bearing mice. Moreover, p-SGP promoted dendritic cells (DCs) maturation and Th1-polarized immune response. Toll-like receptor 4 (TLR4) inhibitor TAK-242 could significantly inhibit the expression of mature molecules on the surface of DCs stimulated by p-SGP, suggesting that p-SGP could play the role of activating DCs through the TLR4 receptor. Results of RNA-seq showed that the Delta-like ligand 4 (DLL4) gene in the pathway Th1 and Th2 cell differentiation was significantly up-regulated in the DCs treated with p-SGP, suggesting that p-SGP has a unique mechanism of enhancing anti-tumor immunity.