RESUMEN
Carbene transfer reactions have emerged as pivotal methodologies for the synthesis of complex molecular architectures. Heme protein-catalyzed carbene transfer reactions have shown promising results on model compounds. However, their limited substrate scope has hindered their application in natural product functionalization. Building upon the foundation of previously published work on a carbene transferase-myoglobin variant, this study employs computer-aided protein engineering to design myoglobin variants, using either docking or the deep learning-based LigandMPNN method. These variants were utilized as catalysts in carbene transfer reactions with a selection of monoterpene substrates featuring C-C double bonds, leading to seven target products. This cost-effective methodology broadens the substrate scope for heme protein-catalyzed reactions, thereby opening novel pathways for research in heme protein functionalities and offering fresh perspectives in the synthesis of bioactive molecules.
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Metano , Monoterpenos , Mioglobina , Mioglobina/química , Metano/química , Metano/análogos & derivados , Monoterpenos/química , Monoterpenos/metabolismo , Ingeniería de Proteínas/métodos , Transferasas/química , Transferasas/metabolismo , Simulación del Acoplamiento MolecularRESUMEN
BACKGROUND: Obese and pre-diabetic women have a higher risk for cardiovascular death than age-matched men with the same symptoms, and there are no effective treatments. We reported that obese and pre-diabetic female Zucker Diabetic Fatty (ZDF-F) rats recapitulate metabolic and cardiac pathology of young obese and pre-diabetic women and exhibit suppression of cardio-reparative AT2R. Here, we investigated whether NP-6A4, a new AT2R agonist with the FDA designation for pediatric cardiomyopathy, mitigate heart disease in ZDF-F rats by restoring AT2R expression. METHODS: ZDF-F rats on a high-fat diet (to induce hyperglycemia) were treated with saline, NP-6A4 (10 mg/kg/day), or NP-6A4 + PD123319 (AT2R-specific antagonist, 5 mg/kg/day) for 4 weeks (n = 21). Cardiac functions, structure, and signaling were assessed by echocardiography, histology, immunohistochemistry, immunoblotting, and cardiac proteome analysis. RESULTS: NP-6A4 treatment attenuated cardiac dysfunction, microvascular damage (-625%) and cardiomyocyte hypertrophy (-263%), and increased capillary density (200%) and AT2R expression (240%) (p < 0.05). NP-6A4 activated a new 8-protein autophagy network and increased autophagy marker LC3-II but suppressed autophagy receptor p62 and autophagy inhibitor Rubicon. Co-treatment with AT2R antagonist PD123319 suppressed NP-6A4's protective effects, confirming that NP-6A4 acts through AT2R. NP-6A4-AT2R-induced cardioprotection was independent of changes in body weight, hyperglycemia, hyperinsulinemia, or blood pressure. CONCLUSIONS: Cardiac autophagy impairment underlies heart disease induced by obesity and pre-diabetes, and there are no drugs to re-activate autophagy. We propose that NP-6A4 can be an effective drug to reactivate cardiac autophagy and treat obesity- and pre-diabetes-induced heart disease, particularly for young and obese women.
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Cardiomiopatías , Cardiopatías , Hiperglucemia , Estado Prediabético , Femenino , Ratas , Animales , Ratas Zucker , Obesidad/complicaciones , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Cardiomiopatías/tratamiento farmacológico , Cardiomiopatías/etiologíaRESUMEN
Wound infection by multidrug-resistant (MDR) bacteria is a major disease burden. Systemic administration of broad-spectrum antibiotics colistin methanesulfonate (CMS) and vancomycin are the last lines of defense against deep wound infections by MDR bacteria. However, systemic administration of CMS and vancomycin are linked to life-threatening vital organ damage. Currently there are no effective topical application strategies to deliver these high molecular weight antibiotics across the stratum corneum. To overcome this difficulty, we tested if high molecular weight antibiotics delivered by Droplette micromist technology device (DMTD), a transdermal delivery device that generates a micromist capable of packaging large molecules, could attenuate deep skin tissue infections. Using green fluorescent protein-tagged E. coli and live tissue imaging, we show that (1) the extent of attenuation of deep-skin E. coli infection was similar when treated with topical DMTD- or systemic IP (intraperitoneal)-delivered CMS; (2) DMTD-delivered micromist did not spread the infection deeper; (3) topical DMTD delivery and IP delivery resulted in similar levels of vancomycin in the skin after a 2 h washout period; and (4) IP-delivered vancomycin was about 1000-fold higher in kidney and plasma than DMTD-delivered vancomycin indicating systemic toxicity. Thus, topical DMTD delivery of these antibiotics is a safe treatment for the difficult-to-treat deep skin tissue infections by MDR bacteria.
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AIM: It is well known that inducing hyperthermia is a type of cancer treatment but some research groups indicate that this treatment is not effective. This article finds and explains the mechanism of this treatment and its possible problems. BACKGROUND: Hyperthermia is commonly known as a state when the temperature of the body rises to a level that can threaten one's health. Hyperthermia is a type of cancer treatment in which body tissue is exposed to high temperatures (up to 45 °C). Research has shown that high temperatures can damage and kill cancer cells, usually with minimal injury to normal tissues. However, this mechanism is not known. MATERIALS AND METHODS: We recently treated cancer cells with different temperatures ranging from 37 °C to 47 °C and further measured their caspase 3 secretion by ELISA, western blot and cell survival rate by microscope. RESULTS: We found that most cancer cells are able to resist hyperthermia more than normal cells most likely via non-activation of caspase3. We also found that hyperthermia-treated (≥41°) cancer cells extend a long pseudopod-like extension in comparison to the same cancer cells under normal conditions. CONCLUSION: Our data here indicates that cancer cells have resistance to higher temperatures compared to normal cells via non-activation of caspase 3. This is a significant issue that needs to be brought to attention as the medical community has always believed that a high temperature treatment can selectively kill cancer/tumor cells. Additionally, we believe that the pseudopod-like extensions of hyperthermia-treated cancer cells must be related to its resistance to hyperthermia.
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Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq). Aq-NF-κB is most similar to NF-κB p100/p105 among vertebrate proteins, with an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile akin to human NF-κB proteins. Like mammalian NF-κB p100, C-terminal truncation allows nuclear translocation of Aq-NF-κB and increases its transcriptional activation activity. Expression of IκB kinases (IKKs) induces proteasome-dependent C-terminal processing of Aq-NF-κB in human cells, and processing requires C-terminal serines in Aq-NF-κB. Unlike NF-κB p100, C-terminal sequences of Aq-NF-κB do not inhibit its DNA-binding activity. Tissue of a black encrusting demosponge contains NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Treatment of sponge tissue with LPS increases both DNA-binding activity and processing of NF-κB. A. queenslandica transcriptomes contain homologs to upstream NF-κB pathway components. This is first functional characterization of NF-κB in sponge, the most basal multicellular animal.
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Secuencia Conservada/genética , Proteínas de Unión al ADN/genética , FN-kappa B/genética , Poríferos/inmunología , Dominios Proteicos/genética , Animales , Proteínas de Unión al ADN/metabolismo , Evolución Molecular , Regulación de la Expresión Génica , FN-kappa B/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
Homogeneous PEGylated lipid bilayer coated highly ordered MSNs (PLMSNs) which were systematically optimized and characterized to co-encapsulate paclitaxel (Tax) and curcumin (Cur) were verified to manifest prolonged and enhanced cytotoxic effect against canine breast cancer cells in our previous study. In this article, we took further study of the pharmacokinetic property, cellular uptake, subcellular localization, in vivo distribution and tumor accumulation ability, and treatment efficacy of the drug delivery system. The results revealed that the delivery system could significantly increase the AUC of two drugs, and the anti-tumor effect showed that both intravenous and intratumoral administration group better controlled the tumor weight than that of other groups (Pâ¯<â¯.05), and the anti-tumor rates were 58.4% and 58.3% respectively. Cell uptake and localization study showed that PLMSNs could effectively carry drugs into cancer cells with sustained release characteristics. The subcellular localization of PLMSNs was mainly in lysosomes and mitochondria. In vivo fluorescence tracing results showed that PLMSNs could be effectively accumulated in the tumor site. The results revealed that the delivery system could effectively reduce the clinical dosage of drugs and reduce its toxic side effects, effectively carry drugs into cancer cells, and exhibit good targeting characteristics for breast cancer.
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Antineoplásicos/farmacocinética , Curcumina/farmacocinética , Membrana Dobles de Lípidos/química , Nanocápsulas/química , Paclitaxel/farmacocinética , Polietilenglicoles/química , Dióxido de Silicio/química , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Transporte Biológico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Curcumina/administración & dosificación , Curcumina/efectos adversos , Perros , Composición de Medicamentos/métodos , Liberación de Fármacos , Quimioterapia Combinada/métodos , Femenino , Humanos , Lisosomas/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/metabolismo , Paclitaxel/administración & dosificación , Paclitaxel/efectos adversos , Porosidad , Ratas Sprague-Dawley , Propiedades de Superficie , Carga Tumoral/efectos de los fármacosRESUMEN
Highly ordered mesoporous silica nanoparticles (MSNs) with pore diameter of 2.754nm and particle size of 115±15nm were prepared with etching method. Homogeneous PEGylated lipid bilayer with 10-15nm thickness was coated around the surface of MSNs using film hydration method. Systematic optimization and characterization of co-encapsulation process of paclitaxel (Tax) and curcumin (Cur) into PEGylated lipid bilayer coated mesoporous silica nanoparticles (PLMSNs) were performed carrying out single factor test, associated with Box-Behnken Design. The concentration of encapsulated drugs was measured by reversed phase high performance liquid chromatography (RP-HPLC) method. Optimal factor settings were as follows: 50mg MSNs, ratio of MSNs to lipid (w/w)=1:1.11, and ratio of lipid to CHO (w/w)=3.93:1. The average experimental EETax, EECur and stability score value were (77.48±2.73) %, (30.70±3.56) % and 4 point respectively based on the conditions mentioned above. Morphology determination of Tax-Cur-PLMSNs revealed that the composite nanoparticles were spherical particals with uniform dispersion. In vitro release experiment indicated that PLMSNs improved dissolution of Tax compared to Tax powder suspension and exhibited sustained release property. Tax-Cur-PLMSNs manifested definite and persistently promoted cytotoxic effect against canine breast cancer cells. This prolonged and enhanced activity of Tax-Cur-PLMSNs might contribute to its sustained release effect.