RESUMEN
BACKGROUND: The SET domain group (SDG) genes encode histone lysine methyltransferases, which regulate gene transcription by altering chromatin structure and play pivotal roles in plant flowering determination. However, few studies have investigated their role in the regulation of flowering in upland cotton. RESULTS: A total of 86 SDG genes were identified through genome-wide analysis in upland cotton (Gossypium hirsutum). These genes were unevenly distributed across 25 chromosomes. Cluster analysis revealed that the 86 GhSDGs were divided into seven main branches. RNA-seq data and qRTâPCR analysis revealed that lysine methyltransferase 3 (KMT3) genes were expressed at high levels in stamens, pistils and other floral organs. Using virus-induced gene silencing (VIGS), functional characterization of GhKMT3;1a and GhKMT3;2a revealed that, compared with those of the controls, the GhKMT3;1a- and GhKMT3;2a-silenced plants exhibited later budding and flowering and lower plant heightwere shorter. In addition, the expression of flowering-related genes (GhAP1, GhSOC1 and GhFT) significantly decreased and the expression level of GhSVP significantly increased in the GhKMT3;1a- and GhKMT3;2a-silenced plants compared with the control plants. CONCLUSION: A total of 86 SDG genes were identified in upland cotton, among which GhKMT3;1a and GhKMT3;2a might regulate flowering by affecting the expression of GhAP1, GhSOC1, GhFT and GhSVP. These findings will provide genetic resources for advanced molecular breeding in the future.
Asunto(s)
Flores , Regulación de la Expresión Génica de las Plantas , Gossypium , N-Metiltransferasa de Histona-Lisina , Proteínas de Plantas , Gossypium/genética , Gossypium/enzimología , Gossypium/fisiología , Flores/genética , Flores/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Genes de Plantas , Silenciador del GenRESUMEN
PURPOSE: A multivesicular liposome (MVL) is a liposomal vehicle designed to achieve sustained release characteristics for drugs with short half-lives. For example, a commercial MVL formulation of bupivacaine has been approved by the U.S. Food and Drug Administration for local and regional analgesia. For complex formulations like those containing MVLs, challenges in developing an in vitro release testing (IVRT) method may hinder generic development and regulatory approval. In this study, we developed an accelerated rotator-based IVRT method with the ability to discriminate bupivacaine MVLs with different quality attributes. METHODS: Three IVRT experimental setups including mesh tube, horizontal shaker, and vertical rotator were screened to ensure that at least 50% of bupivacaine can release from MVLs in 24 h. Sample dilution factors, incubation temperature, and the release media pH were optimized for the IVRT. The reproducibility of the developed IVRT method was validated with commercial bupivacaine MVLs. The discriminative capacity was assessed via comparing commercial and compromised bupivacaine MVL formulations. RESULTS: The rotator-based release setup was chosen due to the capability to obtain 70% of drug release within 24 h. The optimized testing conditions were chosen with a 50-fold dilution factor, a temperature of 37ºC, and a media pH of 7.4. CONCLUSIONS: An accelerated rotator-based IVRT method for bupivacaine MVLs was developed in this study, with the discriminatory ability to distinguish between formulations of different qualities. The developed IVRT method was a robust tool for generic development of MVL based formulations.
Asunto(s)
Bupivacaína , Liposomas , Liberación de Fármacos , Preparaciones de Acción Retardada , Reproducibilidad de los ResultadosRESUMEN
KEY MESSAGE: The silencing of GhGASA14 and the identification of superior allelic variation in its coding region indicate that GhGASA14 may positively regulate flowering and the response to GA3. Gibberellic acid-stimulated Arabidopsis (GASA), a member of the gibberellin-regulated short amino acid family, has been extensively investigated in several plant species and found to be critical for plant growth and development. However, research on this topic in cotton has been limited. In this study, we identified 38 GhGASAs that were dispersed across 18 chromosomes in upland cotton, and all of these genes had a GASA core domain. Transcriptome expression patterns and qRT-PCR results revealed that GhGASA9 and GhGASA14 exhibited upregulated expression not only in the floral organs but also in the leaves of early-maturing cultivars. The two genes were functionally characterized by virus-induced gene silencing (VIGS), and the budding and flowering times after silencing the target genes were later than those of the control (TRV:00). Compared with that in the water-treated group (MOCK), the flowering period of the different fruiting branches in the GA3-treated group was more concentrated. Interestingly, allelic variation was detected in the coding sequence of GhGASA14 between early-maturing and late-maturing accessions, and the frequency of this favorable allele was greater in high-latitude cotton cultivars than in low-latitude ones. Additionally, a significant linear relationship was observed between the expression level of GhGASA14 and flowering time among the 12 upland cotton accessions. Taken together, these results indicated that GhGASA14 may positively regulate flowering time and respond to GA3. These findings could lead to the use of valuable genetic resources for breeding early-maturing cotton cultivars in the future.
Asunto(s)
Flores , Regulación de la Expresión Génica de las Plantas , Giberelinas , Gossypium , Proteínas de Plantas , Gossypium/genética , Gossypium/fisiología , Gossypium/efectos de los fármacos , Flores/genética , Flores/efectos de los fármacos , Flores/fisiología , Flores/crecimiento & desarrollo , Giberelinas/farmacología , Giberelinas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Filogenia , Silenciador del GenRESUMEN
Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.
Asunto(s)
Apolipoproteína A-I , Lipoproteínas HDL , Lipoproteínas HDL/química , Apolipoproteína A-I/farmacología , Apolipoproteína A-I/química , Péptidos/farmacología , Péptidos/química , Colesterol/química , Transporte BiológicoRESUMEN
M10, a novel myricetin derivative, is an anti-inflammatory agent designed for treatment of colitis. Here, we aim to investigate its pharmacokinetic behavior and tissue distribution in a mouse model with colitis. Pharmacokinetics and tissue distribution of M10 and its metabolite myricetin were compared in normal mice and in dextran-sodium-sulfate (DSS)-induced colitis mice. The role of fecal microbiota was also analyzed during metabolism of M10 in vitro. After oral administration, M10 was very low in the plasma of both normal and diseased mice. However, both M10 and myricetin were mainly distributed in the gastrointestinal tract, including the stomach, colon and small intestine, in physiological and pathological conditions. Significantly, M10 and myricetin were found in higher levels in gastrointestinal tracts with inflamed tissues than in normal tissues of mice. An in vitro assay revealed that 80% of M10 was metabolized to myricetin via fecal microbiota. After oral administration, M10 was not absorbed into circulation but mainly distributed in the inflamed submucosal tissues of colitic mice, where it was metabolized into myricetin to prevent colitis development.
Asunto(s)
Colitis Ulcerosa , Colitis , Ratones , Animales , Sulfato de Dextran/efectos adversos , Colitis Ulcerosa/inducido químicamente , Distribución Tisular , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Colon/metabolismo , Modelos Animales de Enfermedad , Sulfatos/metabolismo , Sodio/metabolismo , Ratones Endogámicos C57BLRESUMEN
Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.
Asunto(s)
Lisosomas/enzimología , Fosfolipasas A2/metabolismo , Acilación , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Moleculares , Mutación , Fosfolipasas A2/química , Fosfolipasas A2/genética , Estructura Terciaria de Proteína , Especificidad por SustratoRESUMEN
Lecithin:cholesterol acyltransferase (LCAT) plays a key role in reverse cholesterol transport by transferring an acyl group from phosphatidylcholine to cholesterol, promoting the maturation of high-density lipoproteins (HDL) from discoidal to spherical particles. LCAT is activated through an unknown mechanism by apolipoprotein A-I (apoA-I) and other mimetic peptides that form a belt around HDL. Here, we report the crystal structure of LCAT with an extended lid that blocks access to the active site, consistent with an inactive conformation. Residues Thr-123 and Phe-382 in the catalytic domain form a latch-like interaction with hydrophobic residues in the lid. Because these residues are mutated in genetic disease, lid displacement was hypothesized to be an important feature of apoA-I activation. Functional studies of site-directed mutants revealed that loss of latch interactions or the entire lid enhanced activity against soluble ester substrates, and hydrogen-deuterium exchange (HDX) mass spectrometry revealed that the LCAT lid is extremely dynamic in solution. Upon addition of a covalent inhibitor that mimics one of the reaction intermediates, there is an overall decrease in HDX in the lid and adjacent regions of the protein, consistent with ordering. These data suggest a model wherein the active site of LCAT is shielded from soluble substrates by a dynamic lid until it interacts with HDL to allow transesterification to proceed.
Asunto(s)
Apolipoproteína A-I/fisiología , Fosfatidilcolina-Esterol O-Aciltransferasa/química , Dominio Catalítico , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Activación Enzimática , Humanos , Lipoproteínas HDL/metabolismo , Mutagénesis Sitio-Dirigida , Fosfatidilcolina-Esterol O-Aciltransferasa/metabolismo , Conformación ProteicaRESUMEN
While cancer immunotherapy provides new exciting treatment options for patients, there is an urgent need for new strategies that can synergize with immune checkpoint blockers and boost the patient response rates. We have developed a personalized vaccine nanodisc platform based on synthetic high-density lipoproteins for co-delivery of immunostimulatory agents and tumor antigens, including tumor-specific neoantigens. Here we examined the route of delivery, safety profiles, and therapeutic efficacy of nanodisc vaccination against established tumors. We report that nanodiscs administered via the subcutaneous (SC) or intramuscular (IM) routes were well tolerated in mice without any signs of toxicity. The SC route significantly enhanced nanoparticle delivery to draining lymph nodes, improved nanodisc uptake by antigen-presenting cells, and generated 7-fold higher frequency of neoantigen-specific T cells, compared with the IM route. Importantly, when mice bearing advanced B16F10 melanoma tumors were treated with nanodiscs plus anti-PD-1 and anti-CTLA-4 IgG therapy, the combination immunotherapy exerted potent antitumor efficacy, leading to eradication of established tumors in â¼60% of animals. These results demonstrate nanodiscs customized with patient-specific tumor neoepitopes as a safe and powerful vaccine platform for immunotherapy against advanced cancer.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Antígenos de Neoplasias/administración & dosificación , Vacunas contra el Cáncer/administración & dosificación , Inmunoterapia/métodos , Nanoestructuras/administración & dosificación , Neoplasias/terapia , Vacunación/métodos , Adyuvantes Inmunológicos/uso terapéutico , Animales , Antígenos de Neoplasias/uso terapéutico , Vacunas contra el Cáncer/uso terapéutico , Inyecciones Intramusculares , Inyecciones Subcutáneas , Melanoma Experimental/inmunología , Melanoma Experimental/prevención & control , Ratones , Ratones Endogámicos C57BL , Nanoestructuras/uso terapéutico , Neoplasias/inmunologíaRESUMEN
apoA-I, apoA-I mimetic peptides, and their lipid complexes or reconstituted high-density lipoprotein (HDL) have been studied as treatments for various pathologies. However, consensus is lacking about the best method for administration, by intravenous (IV) or intraperitoneal (IP) routes, and formulation, as an HDL particle or in a lipid-free form. The objective of this study was to systematically examine peptide plasma levels, cholesterol mobilization, and lipoprotein remodeling in vivo following administration of lipid-free apoA-I peptide (22A) or phospholipid reconstituted 22A-sHDL by IV and IP routes. The mean circulation half-life was longer for 22A-sHDL (T1/2 = 6.27 h) than for free 22A (T1/2 = 3.81 h). The percentage of 22A absorbed by the vascular compartment after the IP dosing was â¼50% for both 22A and 22A-sHDL. The strongest pharmacologic response came from IV injection of 22A-sHDL, specifically a 5.3-fold transient increase in plasma-free cholesterol (FC) level compared with 1.3- and 1.8-fold FC increases for 22A-IV and 22A-sHDL-IP groups. Addition of either 22A or 22A-sHDL to rat plasma caused lipoprotein remodeling and appearance of a lipid-poor apoA-I. Hence, both the route of administration and the formulation of apoA-I peptide significantly affect its pharmacokinetics and pharmacodynamics.
Asunto(s)
Apolipoproteína A-I/administración & dosificación , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Péptidos/administración & dosificación , Administración Intravenosa , Animales , Apolipoproteína A-I/metabolismo , Apolipoproteína A-I/farmacocinética , Humanos , Inyecciones Intraperitoneales , Péptidos/metabolismo , Péptidos/farmacocinética , RatasRESUMEN
OBJECTIVES: Recent evidence indicates that high-density lipoprotein (HDL) exerts vasculoprotective activities by promoting activating transcription factor 3 (ATF3), leading to downregulation of toll-like receptor (TLR)-induced inflammatory responses. Systemic lupus erythematosus (SLE) is associated with increased cardiovascular disease risk not explained by the Framingham risk score. Recent studies have indicated oxidised HDL as a possible contributor. We investigated the potential mechanisms by which lupus HDL may lose its anti-inflammatory effects and promote immune dysregulation. METHODS: Control macrophages were challenged with control and SLE HDL in vitro and examined for inflammatory markers by real-time qRT-PCR, confocal microscopy, ELISA and flow cytometry. Lupus-prone mice were treated with an HDL mimetic (ETC-642) in vivo and inflammatory cytokine levels measured by real-time qRT-PCR and ELISA. RESULTS: Compared with control HDL, SLE HDL activates NFκB, promotes inflammatory cytokine production and fails to block TLR-induced inflammation in control macrophages. This failure of lupus HDL to block inflammatory responses is due to an impaired ability to promote ATF3 synthesis and nuclear translocation. This inflammation is dependent on lectin-like oxidised low-density lipoprotein receptor 1 (LOX1R) binding and rho-associated, coiled-coil containing protein kinase 1 and 2 (ROCK1/2) kinase activity. HDL mimetic-treated lupus mice showed significant ATF3 induction and proinflammatory cytokine abrogation. CONCLUSIONS: Lupus HDL promotes proinflammatory responses through NFκB activation and decreased ATF3 synthesis and activity in an LOX1R-dependent and ROCK1/2-dependent manner. HDL mimetics should be explored as potential therapies for inflammation and SLE cardiovascular risk.
Asunto(s)
Factor de Transcripción Activador 3/biosíntesis , Citocinas/genética , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Lupus Eritematoso Sistémico/sangre , ARN Mensajero/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/farmacología , Factor de Transcripción Activador 3/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Amidas/farmacología , Animales , Células Cultivadas , Femenino , Humanos , Macrófagos , Ratones , FN-kappa B/metabolismo , Oxidación-Reducción , Péptidos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Receptores Depuradores de Clase A/genética , Receptores Depuradores de Clase E/genética , Receptores Depuradores de Clase E/metabolismo , Esfingomielinas/farmacología , Bazo/citología , Receptores Toll-Like/metabolismo , Transcripción Genética/efectos de los fármacos , Quinasas Asociadas a rho/metabolismoRESUMEN
Synthetic high-density lipoprotein nanoparticles (sHDL) are a valuable class of nanomedicines with established animal safety profile, clinical tolerability and therapeutic efficacy for cardiovascular applications. In this study we examined how the scavenger receptor B-I-mediated (SR-BI) tumor-targeting ability of sHDL, long plasma circulation half-life, and small particle size (9.6±0.2nm) impacted sHDL accumulation in SR-BI positive colorectal carcinoma cells, 3D tumor spheroids, and in vivo xenografts. We compared tumor accumulation of sHDL with that of liposomes (LIP, 130.7±0.8nm), pegylated liposomes (PEG-LIP, 101±2nm), and pegylated sHDL (12.1±0.1nm), all prepared with the same lipid components. sHDL penetrated deep (210µm) into tumor spheroids and exhibited 12- and 3-fold higher in vivo solid tumor accumulation, compared with LIP (p<0.01) and PEG-LIP (p<0.05), respectively. These results suggest that sHDL with established human safety possess promising intrinsic tumor-targeted properties.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Lipoproteínas HDL/metabolismo , Liposomas/química , Nanopartículas/química , Polietilenglicoles/química , Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Humanos , Tamaño de la Partícula , Receptores Depuradores de Clase B/metabolismo , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
The goal of this study was to understand how the reconstituted HDL (rHDL) phospholipid (PL) composition affects its cholesterol efflux and anti-inflammatory properties. An ApoA-I mimetic peptide, 5A, was combined with either SM or POPC. Both lipid formulations exhibited similar in vitro cholesterol efflux by ABCA1, but 5A-SM exhibited higher ABCG1- and SR-BI-mediated efflux relative to 5A-POPC (P < 0.05). Injection of both rHDLs in rats resulted in mobilization of plasma cholesterol, although the relative potency was 3-fold higher for the same doses of 5A-SM than for 5A-POPC. Formation of preß HDL was observed following incubation of rHDLs with both human and rat plasma in vitro, with 5A-SM inducing a higher extent of preß formation relative to 5A-POPC. Both rHDLs exhibited anti-inflammatory properties, but 5A-SM showed higher inhibition of TNF-α, IL-6, and IL-1ß release than did 5A-POPC (P < 0.05). Both 5A-SM and 5A-POPC showed reduction in total plaque area in ApoE(-/-) mice, but only 5A-SM showed a statistically significant reduction over placebo control and baseline (P < 0.01). The type of PL used to reconstitute peptide has significant influence on rHDL's anti-inflammatory and anti-atherosclerosis properties.
Asunto(s)
Aterosclerosis/metabolismo , Colesterol/metabolismo , Inflamación/metabolismo , Esfingomielinas/metabolismo , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Lipoproteínas HDL/metabolismo , Ratones , Péptidos/administración & dosificación , Fosfatidilcolinas/administración & dosificación , Fosfolípidos/metabolismo , RatasRESUMEN
Exparel is a bupivacaine multivesicular liposomes (MVLs) formulation developed based on the DepoFoam technology. The complex composition and the unique structure of MVLs pose challenges to the development and assessment of generic versions. In the present work, we developed a panel of analytical methods to characterize Exparel with respect to particle size, drug and lipid content, residual solvents, and pH. In addition, an accelerated in vitro drug release assay was developed using a rotator-facilitated, sample-and-separate experimental setup. The proposed method could achieve over 80% of bupivacaine release within 24 h, which could potentially be used for formulation comparison and quality control purposes. The batch-to-batch variability of Exparel was examined by the established analytical methods. Four different batches of Exparel showed good batch-to-batch consistency in drug content, particle size, pH, and in vitro drug release kinetics. However, slight variation in lipid contents were observed.
Asunto(s)
Bupivacaína , Liposomas , Liposomas/química , Preparaciones de Acción Retardada , Liberación de Fármacos , LípidosRESUMEN
ProTide prodrug technology has emerged as a promising way for the development of anti-viral and anti-tumor drugs, whereas, there are fewer applications for the treatment of liver cancer. Herein, a series of distinct 3'-ester ProTide prodrugs of 5-fluoro-2'-deoxyuridine (FdUR) were synthesized and evaluated for their anti-liver cancer activity. The most efficient prodrug 11b reached a sub-micromolar activity (IC50 = 0.42 ± 0.13 µM) against HepG2 and over 100-fold and 200-fold improvements compared to 5-FU, respectively. 11b also demonstrated favorable selectivity towards normal liver cells L-02 (IC50 > 100 µM). In vitro metabolic stability studies revealed that 11b is stable in the plasma and could be activated rapidly in the liver, which supported that 11b is liver-targeted. Importantly, to more accurately evaluate the anti-HCC activity of 11b, the liver orthotopic model was built and 11b significantly suppressed tumor growth (TGI = 75.5%) at a dose of 60 mg/kg/2d in vivo without obvious toxicity. Overall, these promising results indicated that 11b could serve as a safe and effective prodrug of 5-FU nucleoside for liver cancer therapy.
Asunto(s)
Neoplasias Hepáticas , Profármacos , Humanos , Profármacos/farmacología , Desoxiuridina/farmacología , Neoplasias Hepáticas/tratamiento farmacológico , Fluorouracilo/farmacología , Fluorouracilo/uso terapéuticoRESUMEN
Insulin-like growth factor-1 receptor (IGF-1R) has been made an attractive anticancer target due to its overexpression in cancers. However, targeting it has often produced the disappointing results as the role played by cross talk with numerous downstream signalings. Here, we report a disobliging IGF-1R signaling which promotes growth of cancer through triggering the E3 ubiquitin ligase MEX3A-mediated degradation of RIG-I. The active ß-arrestin-2 scaffolds this disobliging signaling to talk with MEX3A. In response to ligands, IGF-1Rß activated the basal ßarr2 into its active state by phosphorylating the interdomain domain on Tyr64 and Tyr250, opening the middle loop (Leu130âCys141) to the RING domain of MEX3A through the conformational changes of ßarr2. The models of ßarr2/IGF-1Rß and ßarr2/MEX3A could interpret the mechanism of the activated-IGF-1R in triggering degradation of RIG-I. The assay of the mutants ßarr2Y64A and ßarr2Y250A further confirmed the role of these two Tyr residues of the interlobe in mediating the talk between IGF-1Rß and the RING domain of MEX3A. The truncated-ßarr2 and the peptide ATQAIRIF, which mimicked the RING domain of MEX3A could prevent the formation of ßarr2/IGF-1Rß and ßarr2/MEX3A complexes, thus blocking the IGF-1R-triggered RIG-I degradation. Degradation of RIG-I resulted in the suppression of the IFN-I-associated immune cells in the TME due to the blockade of the RIG-I-MAVS-IFN-I pathway. Poly(I:C) could reverse anti-PD-L1 insensitivity by recovery of RIG-I. In summary, we revealed a disobliging IGF-1R signaling by which IGF-1Rß promoted cancer growth through triggering the MEX3A-mediated degradation of RIG-I.
RESUMEN
The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rß is translocated into the ER by ß-arrestin2 (ß-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rß. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rß levels. ER IGF-1Rß phosphorylates SERCA2 on Tyr990 to enhance its activity. Mutation of SERCA2-Tyr990 disrupted the interaction of ER IGF-1Rß with SERCA2, and therefore ER IGF-1Rß failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca2+ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rß and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca2+ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr990 in HCC.
RESUMEN
The skeletal muscle fiber profile is closely related to livestock meat quality. However, the molecular mechanisms determining muscle fiber types in donkeys are not completely understood. In this study, we selected the psoas major muscle (PM; mainly composed of oxidative-type muscle fibers) and biceps femoris muscle (BF; mainly composed of glycolytic-type muscle fibers) and systematically compared their mRNA and microRNA transcriptomes via RNA-seq. We identified a total of 2881 differentially expressed genes (DEGs) and 21 known differentially expressed miRNAs (DEmiRs). Furthermore, functional enrichment analysis showed that the DEGs were mainly involved in energy metabolism and actin cytoskeleton regulation. The glycolysis/gluconeogenesis pathway (including up-regulated genes such as PKM, LDHA, PGK1 and ALDOA) was more highly enriched in BF, whereas the oxidative phosphorylation pathway and cardiac muscle contraction (including down-regulated genes such as LDHB, ATP2A2, myosin-7 (MYH7), TNNC1, TPM3 and TNNI1) was more enriched in PM. Additionally, we identified several candidate miRNA-mRNA pairs that might regulate muscle fiber types using the integrated miRNA-mRNA analysis. Combined with the results of protein-protein interaction (PPI) analysis, some interesting DEGs (including ACTN3, TNNT3, TPM2, TNNC2, PKM, TNNC1 and TNNI1) might be potential candidate target genes involved in the miRNA-mediated regulation of the myofibril composition. This study is the first to indicate that DEmiRs, especially eca-miR-193a-5p and eca-miR-370, and potential candidate target genes that are mainly involved in actin binding (e.g., ACTN3, TNNT3 and TNNC1) and the glycolysis/gluconeogenesis pathways (e.g., PKM) might coregulate the myofibril composition in donkeys. This study may provide useful information for improving meat quality traits in Dezhou donkeys.
Asunto(s)
MicroARNs , Transcriptoma , Actinas/metabolismo , Animales , Equidae/genética , Perfilación de la Expresión Génica/métodos , MicroARNs/genética , MicroARNs/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Miosinas/genética , ARN Mensajero/genética , Transcriptoma/genéticaRESUMEN
A liposomal delivery system with a high efficiency of accumulation in tumor tissue and then transportation of the cargo into tumor cells was developed here and evaluated via systemic administration. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)(2000) (DSPE-PEG(2000))-TAT and protective DSPE-PEG(2000) modified liposomes possessing good stability in 50% FBS (fetal bovine serum) and good uptake efficiency were used as the basic formulation (TAT-SL; SL = stealth liposome), and then longer cysteine (Cys)-cleavable PEG(5000) was incorporated to modulate the function of TAT. All of the formulations to be used in vivo had sizes in a range of 80-100 nm and were stable in the presence of 50% FBS. Optical imaging showed that the incorporation of cleavable PEG(5000) into TAT-SL (i.e., C-TAT-SL) led to much more tumor accumulation and much less liver distribution compared with TAT-SL. The in vivo delivery profiles of C-TAT-SL were investigated using DiD as a fluorescent probe. Confocal laser scanning microscopy and flow cytometry showed that C-TAT-SL had a 48% higher (p < 0.001) delivery efficiency in the absence of Cys and a 130% higher (p < 0.001) delivery efficiency in the presence of Cys than the control (SL), indicating the successful targeted delivery of cargo was achieved by C-TAT-SL via systemic administration especially with a subsequent administration of Cys.
Asunto(s)
Antineoplásicos/farmacología , Péptidos de Penetración Celular/química , Sistemas de Liberación de Medicamentos , Productos del Gen tat/química , Liposomas , Neoplasias/tratamiento farmacológico , Polietilenglicoles/química , Animales , Línea Celular Tumoral , Péptidos de Penetración Celular/genética , Modelos Animales de Enfermedad , Portadores de Fármacos/química , Estabilidad de Medicamentos , Colorantes Fluorescentes/química , Productos del Gen tat/genética , Inyecciones Intravenosas , Liposomas/química , Masculino , Ratones , Ratones Endogámicos BALB C , Estructura MolecularRESUMEN
Drug delivery systems to the colon are being actively investigated. However, it is difficult to ensure that an oral preparation disintegrates specifically in the human colon. In this study, a pH- and enzyme-controlled, colon-targeted tablets (PECCTT) was established by using outer pH-coated layer and inner alginate-coated compression layer. The influence of the amount of alginate and enteric coat thickness on drug release had been investigated and the formulation that contained 30% alginate in compression layer and 13% weight gain in pH-coated layer was proved to protect the drug release from stomach and small intestine, the lag time was 7.04 ± 0.17 h, and 84.45 ± 1.3% of prednisone was released at 12 h. The results of drug release behaviors and SEM study indicated that drug release mechanism of PECCTT was corrosion. Hybrid scanner combining SPECT and CT was employed to monitor (99m)Tc-contained tablets in the human gastrointestinal tract (GIT) and to obtain the images of the disintegration process. The results showed that the tablet remained intact during its transit through the upper GIT, the anatomical site of disintegration was found to be the sigmoidal colon, and the disintegration of the tablet started at 8 h post-dose in the volunteer.
Asunto(s)
Alginatos/química , Colon/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Absorción Intestinal/efectos de los fármacos , Animales , Disponibilidad Biológica , Femenino , Humanos , Concentración de Iones de Hidrógeno , Imagen Multimodal/métodos , Tomografía de Emisión de Positrones , Ratas , Comprimidos Recubiertos/química , Tomografía Computarizada por Rayos X , Adulto JovenRESUMEN
Our previous studies found that M10, a myricetin-3-O-ß-d-lactose sodium salt, possessed higher effects of ameliorating ulcerative colitis (UC) than Myricetin in mice. Here, we aim to investigate whether the inhibition of UC is the consequence of the effects of M10 that leads to the changed microbiota. Mice model of UC was induced by dextran sulfate sodium (DSS) treatment. M10 and Myricetin were orally administrated for 12 weeks. We performed 16S rDNA sequencing assay to analyze the composition of gut microbiota isolated from ileocecum. Both M10 and Myricetin normalized the composition of Firmicutes and Actinobacteria as healthy mice had. At genus level, the effects of M10 and Myricetin on colitis were associated to the increase of probiotics, such as Akkermansia, and the inhibition of pathogenic microorganisms, such as Ruminococcus and Parabacteroides. M10 had stronger activity than Myricetin in the improvement of biosynthesis and degradation activities, resulting to increasing metabolism of sulfur, pyruvate, steroid biosynthesis and unsaturated fatty acid biosynthesis in gut. Furthermore, M10 normalized the proportion of Firmicutes and Actinobacteria in gut microbiota. It suggests that the improvements in UC are the consequence of the effect of M10 that leads to the changed intestinal microbiota. Conclusion: M10 contributed the pharmacological effects on UC by modification of the intestinal microbiota.