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Highly efficient resource recycling and comprehensive utilization play a crucial role in achieving the goal of reducing resource wasting, environmental protection, and achieving goal of sustainable development. In this work, the two kinds waste resources of agricultural rice husk and metal ions (Co, Ni, and Mn) from spent lithium-ion batteries have been skillfully utilized to synthesize novel Fenton-like catalysts. Desiliconized rice husk carbon (DRHC) with rich pore structure and large specific surface area from rice husk has been prepared and used as scalable carrier, and dandelion-like nanoparticles cluster could be grown in situ on the surface of the carrier by using metal ions contained waste water. The designed catalysts (X@DRHC) as well as their preparation process were characterized in detail by SEM, TEM, BET, XRD and XPS, respectively. Meanwhile, their catalytic abilities were also studied by activating potassium peroxomonosulfate (PMS) to remove methylene blue (MB). The results indicate X@DRHC displays excellent degradation efficiency on MB with wide pH range and stable reusability, which is suitable for the degradation of various dyes. This work has realized the recycling and high-value utilization of waste resources from biomass and spent lithium-ion batteries, which not only creates an efficient way to dispose waste resources, but also shows high economic benefits in large-scale water treatment.
Assuntos
Lítio , Oryza , Peróxidos , Carbono , Metais , Reciclagem/métodos , Fontes de Energia Elétrica , ÍonsRESUMO
Microtubule-associated protein tau is essential for microtubule assembly and stabilization. Hyperphosphorylation of the microtubule-associated protein tau plays an important pathological role in the development of Alzheimer's disease and other tauopathies. In vivo studies using kinase inhibitors suggest that reducing tau phosphorylation levels has therapeutic potential; however, such approaches showed limited benefits. We sought to further develop our phosphorylation targeting chimera (PhosTAC) technology to specifically induce tau dephosphorylation. Herein, we use small molecule-based PhosTACs to recruit tau to PP2A, a native tau phosphatase. PhosTACs induced the formation of a stable ternary complex, leading to rapid, efficient, and sustained tau dephosphorylation, which also correlated with the enhanced downregulation of tau protein. Mass spectrometry data validated that PhosTACs downregulated multiple phosphorylation sites of tau. We believe that PhosTAC possesses several advantages over current strategies to modulate tau phosphorylation and represents a new avenue for disease-modifying therapies for tauopathies.
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Proteolysis-targeting chimeras (PROTACs), an emerging paradigm-shifting technology, hijacks the ubiquitin-proteasome system for targeted protein degradation. PROTACs induce ternary complexes between an E3 ligase and POI, and this induced proximity leads to polyUb chain formation on substrates and eventual proteasomal-mediated POI degradation. PROTACs have shown great therapeutic potential by degrading many disease-causing proteins, such as the androgen receptor and BRD4. The PROTAC technology has advanced significantly in the last two decades, with the repertoire of PROTAC targets increased tremendously. Herein, we describe recent developments of PROTAC technology, focusing on mechanistic and kinetic studies, pharmacokinetic study, spatiotemporal control of PROTACs, covalent PROTACs, resistance to PROTACs, and new E3 ligands.
Assuntos
Proteínas/metabolismo , Células HeLa , Humanos , Imunoconjugados/metabolismo , Cinética , Ligantes , Luz , Conformação Proteica , Proteínas/química , Proteólise , Fatores de Transcrição/metabolismoRESUMO
Selenium (Se), an essential component of deiodinases (DIOs), regulates the contents of thyroid hormones and thus improves animal growth. To explore the influences of selenium supplementation on fish growth metabolism, a total of 270 healthy grass carp (Ctenopharyngodon idella) were divided into three groups and feed three graded dietary selenium (0.141, 0.562, and 1.044 mg Se/kg) levels. The results showed that after 60-day feeding, dietary selenium improved the final body weight and specific growth rate (SGR) of grass carp. The hepatic DIO activities in selenium-supplemented groups were higher than those in control group. A significant increase in triiodothyronine (T3), free triiodothyronine (FT3), and thyroid-stimulating hormone (TSH) levels was accompanied by a decrease in the contents of thyroxine (T4) and free thyroxine (FT4) in selenium-supplemented groups. The histopathological observation of thyroid suggested that selenium deficiency resulted in hypertrophy of follicular epithelial cells. Moreover, the gene relative expression levels of dio1, dio2, and dio3 showed an increasing trend with the rising concentration of dietary selenium. The transcription levels of HPT axis-related genes (crh, tsh-ß, ttr, tr-s, tpo, nis) and GH/IGF1-related genes (gh, ghr, igf1, igf1r) were significantly upregulated in selenium-supplemented groups. No significant differences in the above indicators were observed between 0.562 and 1.044 mg Se/kg diet group except T3 content and dio1 relative expression ratio. These results indicate that dietary selenium supplementation improves the hepatic DIO activities and thyroid hormone metabolism and regulates the transcription levels of HPT and GH/IGF axis-related genes, which may be responsible for the growth promotion in grass carp.
Assuntos
Carpas , Suplementos Nutricionais , Selênio/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Carpas/sangue , Carpas/crescimento & desenvolvimento , Carpas/metabolismo , Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento/genética , Hipotálamo , Fator de Crescimento Insulin-Like I/genética , Iodeto Peroxidase/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hipófise , Receptor IGF Tipo 1/genética , Receptores da Somatotropina/genética , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/patologia , Tireotropina/sangue , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
Endosomal Toll-like receptors (TLR3, TLR7, TLR8, and TLR9) are highly analogous sensors for various viral or bacterial RNA and DNA molecular patterns. Nonetheless, few small molecules can selectively modulate these TLRs. In this manuscript, we identified the first human TLR8-specific small-molecule antagonists via a novel inhibition mechanism. Crystal structures of two distinct TLR8-ligand complexes validated a unique binding site on the protein-protein interface of the TLR8 homodimer. Upon binding to this new site, the small-molecule ligands stabilize the preformed TLR8 dimer in its resting state, preventing activation. As a proof of concept of their therapeutic potential, we have demonstrated that these drug-like inhibitors are able to suppress TLR8-mediated proinflammatory signaling in various cell lines, human primary cells, and patient specimens. These results not only suggest a novel strategy for TLR inhibitor design, but also shed critical mechanistic insight into these clinically important immune receptors.
Assuntos
Bibliotecas de Moléculas Pequenas/farmacologia , Receptor 8 Toll-Like/antagonistas & inibidores , Artrite Reumatoide/imunologia , Sítios de Ligação , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Imunidade Inata , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ligantes , Modelos Moleculares , Multimerização Proteica , Estabilidade Proteica , Bibliotecas de Moléculas Pequenas/química , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/imunologia , Receptor 8 Toll-Like/genética , Receptor 8 Toll-Like/imunologia , TransfecçãoRESUMO
This paper investigates the impact of online interaction between investors and enterprises on stock liquidity, using data from A-share listed companies in China from 2010 to 2021. Firstly, our findings reveal that more frequent interaction leads to better stock liquidity, and this result remains consistent across various robustness tests. Secondly, we observe that the expected tenure of senior executives and the ratio of institutional investor ownership exert a significant moderating effect on this relationship. Thirdly, this effect varies across enterprises at different development stages and with different ownership structures, being more pronounced in growing and privately-owned companies. Furthermore, this paper finds an inverted U-shaped relationship between reply length and stock liquidity, indicating that excessively long replies may introduce noise and negatively affect liquidity. This study provides new insights into how online interactions can improve market efficiency and offers practical implications for corporate governance and investor relations.
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Internet , Investimentos em Saúde , China , Humanos , Propriedade , Comércio , População do Leste AsiáticoRESUMO
We recently developed a heterobifunctional approach [phosphorylation targeting chimeras (PhosTACs)] to achieve the targeted protein dephosphorylation (TPDephos). Here, we envisioned combining the inhibitory effects of receptor tyrosine kinase inhibitors (RTKIs) and the active dephosphorylation by phosphatases to achieve dual inhibition of kinases. We report an example of tyrosine phosphatase-based TPDephos and the effective epidermal growth factor receptor (EGFR) tyrosine dephosphorylation. We also used phosphoproteomic approaches to study the signaling transductions affected by PhosTAC-related molecules at the proteome-wide level. This work demonstrated the differential signaling pathways inhibited by PhosTAC compared with the TKI, gefitinib. Moreover, a covalent PhosTAC selective for mutated EGFR was developed and showed its inhibitory potential for dysregulated EGFR. Last, EGFR PhosTACs, consistent with EGFR dephosphorylation profiles, induced apoptosis and inhibited cancer cell viability during prolonged PhosTAC treatment. PhosTACs showcased their potential of modulating RTKs activity, expanding the scope of bifunctional molecule utility.
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Receptores ErbB , Quimera de Direcionamento de Proteólise , Apoptose , Linhagem Celular Tumoral , Fosforilação , Transdução de Sinais , Tirosina/metabolismo , Humanos , Quimera de Direcionamento de Proteólise/metabolismoRESUMO
Under unfavorable agricultural conditions, ammonia toxicity has become a major problem, resulting in a large number of deaths. Ammonia has been shown to be hepatotoxic. Research has also shown that ammonia can damage the livers of carp, but the mechanism is unclear. In this study, normal grass carp hepatocytes (L8824) were exposed to ammonia water to investigate the effect of ammonia on hepatocyte injury and apoptosis and its mechanism. The results showed that ammonia (50 mM) reduced the viability of L8824 cells and increased glutamic pyruvic transaminase (ALT, up 144.95%, P < 0.01) and glutamic oxalacetic transaminase (AST, up 65.27%, P < 0.01). Furthermore, exposure to ammonia induced oxidative stress and endoplasmic reticulum (ER) stress in L8824 cells. Elevated levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased mitochondrial membrane potential indicated that L8824 cells suffered oxidative damage. Endoplasmic reticulum stress manifests as increased expression degrees of PERK, ATF4, and IRE-1α. These results confirmed the toxicity of ammonia to hepatocytes. In addition, the rate of apoptosis in L8824 cells was increased 69.66% after exposure to ammonia (50 mM, P < 0.01). However, pretreatment of L8824 cells with ER stress inhibitor 2-APB reduced ammonia-induced calcium release (26.50%, P < 0.01) in endoplasmic reticulum. These results indicate that ammonia can exert toxic effects on L8824 cells through inducing endoplasmic reticulum stress and oxidative stress, resulting in apoptosis in L8824 cells.
Assuntos
Compostos de Amônio , Carpas , Animais , Compostos de Amônio/metabolismo , Amônia/metabolismo , Carpas/metabolismo , Fígado , Estresse Oxidativo , Hepatócitos , Estresse do Retículo Endoplasmático , Linhagem Celular , Espécies Reativas de Oxigênio/metabolismo , ApoptoseRESUMO
Phosphorylation is one of the most important post-translational modifications (PTMs) of proteins, governing critical protein functions. Most human proteins have been shown to undergo phosphorylation, and phosphoproteomic studies have been widely applied due to recent advancements in high-resolution mass spectrometry technology. Although the experimental workflow for phosphoproteomics has been well-established, it would be useful to optimize and summarize a detailed, feasible protocol that combines phosphoproteomics and data-independent acquisition (DIA), along with follow-up data analysis procedures due to the recent instrumental and bioinformatic advances in measuring and understanding tens of thousands of site-specific phosphorylation events in a single experiment. Here, we describe an optimized Phos-DIA protocol, from sample preparation to bioinformatic analysis, along with practical considerations and experimental configurations for each step. The protocol is designed to be robust and applicable for both small-scale phosphoproteomic analysis and large-scale quantification of hundreds of samples for studies in systems biology and systems medicine.
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The serine/threonine kinase AKT is a central node in cell signaling. While aberrant AKT activation underlies the development of a variety of human diseases, how different patterns of AKT-dependent phosphorylation dictate downstream signaling and phenotypic outcomes remains largely enigmatic. Herein, we perform a systems-level analysis that integrates methodological advances in optogenetics, mass spectrometry-based phosphoproteomics, and bioinformatics to elucidate how different intensity, duration, and pattern of Akt1 stimulation lead to distinct temporal phosphorylation profiles in vascular endothelial cells. Through the analysis of ~35,000 phosphorylation sites across multiple conditions precisely controlled by light stimulation, we identify a series of signaling circuits activated downstream of Akt1 and interrogate how Akt1 signaling integrates with growth factor signaling in endothelial cells. Furthermore, our results categorize kinase substrates that are preferably activated by oscillating, transient, and sustained Akt1 signals. We validate a list of phosphorylation sites that covaried with Akt1 phosphorylation across experimental conditions as potential Akt1 substrates. Our resulting dataset provides a rich resource for future studies on AKT signaling and dynamics.
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Células Endoteliais , Proteínas Proto-Oncogênicas c-akt , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Endoteliais/metabolismo , Optogenética , Transdução de Sinais , Proteínas Serina-Treonina Quinases/metabolismo , FosforilaçãoRESUMO
Toll-like receptor 8 (TLR8) is a pattern recognition receptor that senses RNA degradation products and initiates immune responses. TLR8 overactivation is associated with autoimmune diseases. Herein, we describe the evaluation and validation of TLR8 antagonists in HEK-Blue cells via secreted embryonic alkaline phosphatase (SEAP) assay, WST assay, ITC and immunoblotting. These assays can facilitate the development of TLR8 antagonists; this protocol can also be adapted to analyze agonists and antagonists for other TLRs. For complete details on the use and execution of this protocol, please refer to Hu et al. (2018).
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Fosfatase Alcalina , Receptor 8 Toll-Like , Linhagem Celular , Humanos , Receptor 8 Toll-Like/antagonistas & inibidoresRESUMO
The present study was conducted to investigate the protective effects of selenium on the oxidative damage of kidney cells (CIK) caused by nitrite exposure in grass carp (Ctenopharyngodon idella). Cells were pre-incubated by Na2SeO3 (10 µmol/L) for 12 h and then exposed to NaNO2 (25 mg/L) for 24 h, the cell viability, apoptosis, gene expression, and antioxidant enzyme activity were assayed. The results show that nitrite reduced cell viability and induced apoptosis, and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the sod, cat, and gpx genes reduced (p < 0.05), while the intracellular calcium ion concentration increased (p < 0.05). Interestingly, selenium treatment significantly alleviated the nitrite induced changes in cell growth, apoptosis, and calcium influx. The cell viability after low-concentration selenium treatment is higher than that of normal cells (p < 0.05). CIK cells were pre-incubated with Na2SeO3 and then exposed to NaNO2, the antioxidant indicators could be maintained at normal levels. And compared with nitrite exposure, intracellular calcium ion concentration and apoptotic rate of selenium-incubated still decreased. The expressions of Nrf2 and Keap1 genes increased significantly in CIK cells treated with sodium selenite for 12 h, and the same trend as the enzyme activities of this group. The results show that the supplement of selenium can enhance the cell's resistance to sodium nitrite exposure to a certain extent, by alleviating the antioxidant imbalance, high apoptosis rate, and intracellular calcium ion disturbance caused by nitrite exposure. And the Nrf2-Keap1 pathway may play an important role in the process.
Assuntos
Carpas , Selênio , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Cálcio/metabolismo , Carpas/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Rim/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Selênio/metabolismo , Selênio/farmacologia , Nitrito de Sódio/metabolismo , Nitrito de Sódio/farmacologia , Superóxido Dismutase/metabolismoRESUMO
Hemiculter leucisculus is an invasive fish and widely distributed in the Xinjiang Tarim River. In this study, RAD-seq was used to explore the genetic diversity and population subgroup structure of H. leucisculus in the Tarim River and develop relevant Simple Sequence Repeat (SSR) markers. The study collected 40 samples distributed at four different sites of the Tarim River. A total of 7,291,260 single nucleotide polymorphisms (SNPs) were obtained. The genetic diversity results showed that the population genetic diversity level of H. leucisculus was low. The population pairwise FST values ranged from 0.231 to 0.258, indicating that there was moderate genetic differentiation among these populations. AMOVA showed that the genetic variation within populations accounted for 92.31% of the total variation. The principal component analysis (PCA) and neighbor joining (NJ) tree revealed that the four populations could be separated into two clusters (upper-middle and downstream populations) and the individuals from Taitema Lake (TTMH) showed differences and had a bigger geographic distance than the others. There is the probability that the H. leucisculus from Bosten Lake entered Taitema Lake to breed and then expanded into the Tarim River due to the water diversion projects in location. In addition, 147,705 SSRs loci were detected and 22,651 SSR primer pairs were developed. This study will contribute to providing valuable molecular data for the management of wild populations, marker-assisted selection and resource exploitation of H. leucisculus.
Assuntos
Cipriniformes , Repetições de Microssatélites , Animais , Repetições de Microssatélites/genética , Rios , Variação Genética/genética , ÁguaRESUMO
Protein phosphorylation, which regulates many critical aspects of cell biology, is dynamically governed by kinases and phosphatases. Many diseases are associated with dysregulated hyperphosphorylation of critical proteins, such as retinoblastoma protein in cancer. Although kinase inhibitors have been widely applied in the clinic, growing evidence of off-target effects and increasing drug resistance prompts the need to develop a new generation of drugs. Here, we propose a proof-of-concept study of phosphorylation targeting chimeras (PhosTACs). Similar to PROTACs in their ability to induce ternary complexes, PhosTACs focus on recruiting a Ser/Thr phosphatase to a phosphosubstrate to mediate its dephosphorylation. However, distinct from PROTACs, PhosTACs can uniquely provide target gain-of-function opportunities to manipulate protein activity. In this study, we applied a chemical biology approach to evaluate the feasibility of PhosTACs by recruiting the scaffold and catalytic subunits of the PP2A holoenzyme to protein substrates such as PDCD4 and FOXO3a for targeted protein dephosphorylation. For FOXO3a, this dephosphorylation resulted in the transcriptional activation of a FOXO3a-responsive reporter gene.
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Quimera/metabolismo , Fosfoproteínas/química , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Reguladoras de Apoptose , Domínio Catalítico , Ativação Enzimática , Proteína Forkhead Box O3 , Células HeLa , Holoenzimas/química , Humanos , Fosforilação , Proteínas de Ligação a RNA , Relação Estrutura-AtividadeRESUMO
Many diseases, including cancer, stem from aberrant activation or overexpression of oncoproteins that are associated with multiple signaling pathways. Although proteins with catalytic activity can be successfully drugged, the majority of other protein families, such as transcription factors, remain intractable due to their lack of ligandable sites. In this study, we report the development of TRAnscription Factor TArgeting Chimeras (TRAFTACs) as a generalizable strategy for targeted transcription factor degradation. We show that TRAFTACs, which consist of a chimeric oligonucleotide that simultaneously binds to the transcription factor of interest (TOI) and to HaloTag-fused dCas9 protein, can induce degradation of the former via the proteasomal pathway. Application of TRAFTACs to two oncogenic TOIs, NF-κB and brachyury, suggests that TRAFTACs can be successfully employed for the targeted degradation of other DNA-binding proteins. Thus, TRAFTAC technology is potentially a generalizable strategy to induce degradation of other transcription factors both in vitro and in vivo.
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Oligonucleotídeos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Humanos , Peixe-ZebraRESUMO
We recently showed that TLR8 is critical for the detection of Gram-positive bacteria by human monocytes. Here, we hypothesized that TLR8 and complement together regulate antibacterial responses in human blood. Anticoagulated blood was treated with selective inhibitors of TLR8 and/or complement C5, and then challenged with live Streptococcus agalactiae (Group B streptococcus, GBS), Staphylococcus aureus, or Escherichia coli. Cytokine production, plasma membrane permeability, bacterial survival, phagocytosis, and activation of coagulation was examined. GBS and S. aureus, but not E. coli, triggered TLR8-dependent production of IL-12p70, IL-1ß, TNF, and IL-6 in fresh human whole blood. In purified polymorphonuclear neutrophils (PMN), GBS and S. aureus induced IL-8 release in part via TLR8, whereas PMN plasma membrane leakage and extracellular DNA levels increased independently of TLR8. TLR8 was more important than C5 for bacteria-induced production of IL-12p70, IL-1ß, and TNF in blood, whereas IL-8 release was more C5 dependent. Both TLR8 and C5 induced IL-6 release and activation of prothrombin cleavage, and here their combined effects were additive. Blocking of C5 or C5aR1 attenuated phagocytosis and increased the extracellular growth of GBS in blood, whereas TLR8 inhibition neither reduced phagocytosis nor intracellular killing of GBS and S. aureus. In conclusion, TLR8 is more important than C5 for production of IL-12p70, IL-1ß, and TNF upon GBS and S. aureus infection in blood, whereas C5 is central for IL-8 release and phagocytosis. Both TLR8 and C5 mediate IL-6 release and activation of coagulation during challenge with Gram-positive bacteria in blood.
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Complemento C5/metabolismo , Citocinas/sangue , Bactérias Gram-Positivas/fisiologia , Trombina/metabolismo , Receptor 8 Toll-Like/sangue , Coagulação Sanguínea , Membrana Celular/metabolismo , Sobrevivência Celular , DNA/metabolismo , Humanos , Interleucina-8/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Viabilidade Microbiana , Monócitos/metabolismo , Neutrófilos/metabolismo , Receptor 8 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/metabolismoRESUMO
During HIV infection, cell-to-cell transmission results in endosomal uptake of the virus by target CD4+ T cells and potential exposure of the viral ssRNA genome to endosomal Toll-like receptors (TLRs). TLRs are instrumental in activating inflammatory responses in innate immune cells, but their function in adaptive immune cells is less well understood. Here we show that synthetic ligands of TLR8 boosted T cell receptor signaling, resulting in increased cytokine production and upregulation of surface activation markers. Adjuvant TLR8 stimulation, but not TLR7 or TLR9, further promoted T helper cell differentiation towards Th1 and Th17. In addition, we found that endosomal HIV induced cytokine secretion from CD4+ T cells in a TLR8-specific manner. TLR8 engagement also enhanced HIV-1 replication and potentiated the reversal of latency in patient-derived T cells. The adjuvant TLR8 activity in T cells can contribute to viral dissemination in the lymph node and low-grade inflammation in HIV patients. In addition, it can potentially be exploited for therapeutic targeting and vaccine development.
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Infecções por HIV/imunologia , HIV-1/imunologia , Células Th1/imunologia , Células Th17/imunologia , Receptor 8 Toll-Like/metabolismo , Linhagem Celular , Infecções por HIV/transmissão , Humanos , Imunidade Inata/imunologia , Transdução de Sinais/imunologia , Receptor 8 Toll-Like/imunologiaRESUMO
TLR8 is an endosomal sensor of RNA degradation products in human phagocytes, and is involved in the recognition of viral and bacterial pathogens. We previously showed that in human primary monocytes and monocyte derived macrophages, TLR8 senses entire Staphylococcus aureus and Streptococcus agalactiae (group B streptococcus, GBS), resulting in the activation of IRF5 and production of IFNß, IL-12p70, and TNF. However, the quantitative and qualitative impact of TLR8 for the sensing of bacteria have remained unclear because selective inhibitors have been unavailable. Moreover, while we have shown that TLR2 activation attenuates TLR8-IRF5 signaling, the molecular mechanism of this crosstalk is unknown. We here used a recently developed chemical antagonist of TLR8 to determine its role in human primary monocytes challenged with S. aureus, GBS, Streptococcus pneumonia, Pseudomonas aeruginosa, and E. coli. The inhibitor completely blocked cytokine production in monocytes stimulated with TLR8-agonists, but not TLR2-, and TLR4-agonists. Upon challenge with S. aureus, GBS, and S. pneumonia, the TLR8 inhibitor almost eliminated the production of IL-1ß and IL-12p70, and it strongly reduced the release of IL-6, TNF, and IL-10. With P. aeruginosa infection, the TLR8 inhibitor impaired the production of IL-12p70 and IL-1ß, while with E. coli infection the inhibitor had less effect that varied depending on the strain and conditions. Signaling via TLR2, TLR4, or TLR5, but not TLR8, rapidly eliminated IRAK-1 detection by immunoblotting due to IRAK-1 modifications during activation. Silencing of IRAK-1 reduced the induction of IFNß and TNF by TLR8 activation, suggesting that IRAK-1 is required for TLR8-IRF5 signaling. The TLR-induced modifications of IRAK-1 also correlated closely with attenuation of TLR8-IRF5 activation, suggesting that sequestration and/or modification of Myddosome components by cell surface TLRs limit the function of TLR8. Accordingly, inhibition of CD14- and TLR4-activation during E. coli challenge increased the activation of IRF5 and the production of IL-1ß and IL-12p70. We conclude that TLR8 is a dominating sensor of several species of pyogenic bacteria in human monocytes, while some bacteria attenuate TLR8-signaling via cell surface TLR- activation. Taken together, TLR8 appears as a more important sensor in the antibacterial defense system than previously known.
Assuntos
Macrófagos/imunologia , Monócitos/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Streptococcus agalactiae/fisiologia , Receptor 8 Toll-Like/metabolismo , Células Cultivadas , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon beta/metabolismo , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-12/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Rational design of drug-like small-molecule ligands based on structural information of proteins remains a significant challenge in chemical biology. In particular, designs targeting protein-protein interfaces have met little success given the dynamic nature of the protein surfaces. Herein, we utilized the structure of a small-molecule ligand in complex with Toll-like receptor 8 (TLR8) as a model system due to TLR8's clinical relevance. Overactivation of TLR8 has been suggested to play a prominent role in the pathogenesis of various autoimmune diseases; however, there are still few small-molecule antagonists available, and our rational designs led to the discovery of six exceptionally potent compounds with â¼picomolar IC50 values. Two X-ray crystallographic structures validated the contacts within the binding pocket. A variety of biological evaluations in cultured cell lines, human peripheral blood mononuclear cells, and splenocytes from human TLR8-transgenic mice further demonstrated these TLR8 inhibitors' high efficacy, suggesting strong therapeutic potential against autoimmune disorders.
Assuntos
Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Receptor 8 Toll-Like/antagonistas & inibidores , Animais , Doenças Autoimunes/tratamento farmacológico , Sítios de Ligação , Células Cultivadas , Desenho Assistido por Computador , Cristalografia por Raios X , Desenho de Fármacos , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Receptor 8 Toll-Like/química , Receptor 8 Toll-Like/metabolismoRESUMO
Water-in-oil-in-water (w/o/w) double emulsions are potential for enhancing oral bioavailability of drugs with high solubility and low permeability, but their industrial application is limited due to the instability. Herein, we developed a novel formulation, self-double-emulsifying drug delivery systems (SDEDDS) by formulating mixtures of hydrophilic surfactants and water-in-oil (w/o) emulsions, which were easier to be stable through formulations optimization. SDEDDS can spontaneously emulsify to water-in-oil-in-water (w/o/w) double emulsions in the mixed aqueous gastrointestinal environment, with drugs encapsulated in the internal water phase of the double emulsions. We employed SDEDDS to improve the oral absorption of pidotimod, a peptide-like drug with high solubility and low permeability. The optimized pidotimod-SDEDDS were found to be stable up to 6 months under 25°C. Plasma concentration-time profiles from pharmacokinetic studies in rats dosed with SDEDDS showed 2.56-fold (p<0.05) increased absorption of pidotimod, compared to the pidotimod solution. Histopathologic studies confirmed that SDEDDS exerted absorption promoting effect without serious local damages. These studies demonstrate that SDEDDS may be a promising strategy for peroral delivery of peptide and peptidomimetic drugs.