RESUMEN
Protein arginine N-methyltransferases are a family of epigenetic enzymes responsible for monomethylation or dimethylation of arginine residues on histones. Dysregulation of protein arginine N-methyltransferase activity can lead to aberrant gene expression and cancer. Recent studies have shown that PRMT2 expression and histone H3 methylation at arginine 8 are correlated with disease severity in glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma. In this study, we explore a noncatalytic mechanistic role for PRMT2 in histone methylation by investigating interactions between PRMT2, histone peptides and proteins, and other PRMTs using analytical and enzymatic approaches. We quantify interactions between PRMT2, peptide ligands, and PRMT1 in a cofactor- and domain-dependent manner using differential scanning fluorimetry. We found that PRMT2 modulates the substrate specificity of PRMT1. Using calf thymus histones as substrates, we saw that a 10-fold excess of PRMT2 promotes PRMT1 methylation of both histone H4 and histone H2A. We found equimolar or a 10-fold excess of PRMT2 to PRMT1 can improve the catalytic efficiency of PRMT1 towards individual histone substrates H2A, H3, and H4. We further evaluated the effects of PRMT2 towards PRMT1 on unmodified histone octamers and mononucleosomes and found marginal PRMT1 activity improvements in histone octamers but significantly greater methylation of mononucleosomes in the presence of 10-fold excess of PRMT2. This work reveals the ability of PRMT2 to serve a noncatalytic role through its SH3 domain in driving site-specific histone methylation marks.
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Histonas , Proteína-Arginina N-Metiltransferasas , Arginina/metabolismo , Histonas/metabolismo , Metilación , Proteína-Arginina N-Metiltransferasas/metabolismo , Fluorometría , Especificidad por Sustrato , Estabilidad Proteica , Unión Proteica , Dominios Proteicos , Ligandos , HumanosRESUMEN
Lilii Bulbus is a folk medicine for both culinary and medicinal purpose. In traditional medicine theory, Lilii Bulbus is usually used as an complementary therapy for nourishing the heart and lung, clearing heat in the treatment of mental instability and depression. In this study, NLPS-1a (Mw = 2610 Da, DP = 16), a water-soluble non-starch Lilii Bulbus polysaccharides, was isolated and purified. Structural analysis showed that NLPS-1a mainly contained Man and Glc with a molar ratio of 11.137 and 9.427. The glycosidic linkages of NLPS-1a were 1,3-Manp (59.93%), 1,2-Glcp (37.93%), T-Glcp (1.21%) and T-Manp (0.93%), indicating the highly-linear structures. In addition, NLPS-1a could significantly repair the injury of PC12 cells induced by corticosterone (CORT), reduce Lactate dehydrogenase (LDH) leakage and decrease the cell apoptosis in a dose-dependent manner. Above all, the results indicated that NLPS-1a had protective effects against CORT-induced neurotoxicity in PC12 cells, and might be a natural antidepressant, which enriched the study of the metabolic mechanism between herbal polysaccharides and antidepressant.
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Apoptosis , Corticosterona , Ratas , Animales , Humanos , Corticosterona/toxicidad , Células PC12 , Polisacáridos/farmacología , Antidepresivos/farmacologíaRESUMEN
BACKGROUND: West Nile virus (WNV) is a rapidly spreading mosquito-borne virus accounted for neuroinvasive diseases. An insight into WNV-host factors interaction is necessary for development of therapeutic approaches against WNV infection. CD11b has key biological functions and been identified as a therapeutic target for several human diseases. The purpose of this study was to determine whether CD11b was implicated in WNV infection. METHODS: SH-SY5Y cells with and without MEK1/2 inhibitor U0126 or AKT inhibitor MK-2206 treatment were infected with WNV. CD11b mRNA levels were assessed by real-time PCR. WNV replication and expression of stress (ATF6 and CHOP), pro-inflammatory (TNF-α), and antiviral (IFN-α, IFN-ß, and IFN-γ) factors were evaluated in WNV-infected SH-SY5Y cells with CD11b siRNA transfection. Cell viability was determined by MTS assay. RESULTS: CD11b mRNA expression was remarkably up-regulated by WNV in a time-dependent manner. U0126 but not MK-2206 treatment reduced the CD11b induction by WNV. CD11b knockdown significantly decreased WNV replication and protected the infected cells. CD11b knockdown markedly increased TNF-α, IFN-α, IFN-ß, and IFN-γ mRNA expression induced by WNV. ATF6 mRNA expression was reduced upon CD11b knockdown following WNV infection. CONCLUSION: These results demonstrate that CD11b is involved in maintaining WNV replication and modulating inflammatory as well as antiviral immune response, highlighting the potential of CD11b as a target for therapeutics for WNV infection.
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Antígeno CD11b , Replicación Viral , Virus del Nilo Occidental , Humanos , Replicación Viral/efectos de los fármacos , Virus del Nilo Occidental/fisiología , Virus del Nilo Occidental/inmunología , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Línea Celular Tumoral , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virología , Neuroblastoma/inmunología , Neuroblastoma/virología , Interacciones Huésped-Patógeno/inmunología , Supervivencia Celular/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Recombinant adeno-associated virus (rAAV) is a commonly used in vivo gene therapy vector because of its nonpathogenicity, long-term transgene expression, broad tropism, and ability to transduce both dividing and nondividing cells. However, rAAV vector production via transient transfection of mammalian cells typically yields a low fraction of filled-to-total capsids (~1%-30% of total capsids produced). Analysis of our previously developed mechanistic model for rAAV2/5 production attributed these low fill fractions to a poorly coordinated timeline between capsid synthesis and viral DNA replication and the repression of later phase capsid formation by Rep proteins. Here, we extend the model by quantifying the expression dynamics of total Rep proteins and their influence on the key steps of rAAV2/5 production using a multiple dosing transfection of human embryonic kidney 293 (HEK293) cells. We report that the availability of preformed empty capsids and viral DNA copies per cell are not limiting to the capsid-filling reaction. However, optimal expression of Rep proteins (<240 ± 13 ag per cell) enables enrichment of the filled capsid population (>12% of total capsids/cell) upstream. Our analysis suggests increased enrichment of filled capsids via regulating the expression of Rep proteins is possible but at the expense of per cell capsid titer in a triple plasmid transfection. Our study reveals an intrinsic limitation of scaling rAAV2/5 vector genome (vg) production and underscores the need for approaches that allow for regulating the expression of Rep proteins to maximize vg titer per cell upstream.
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Pyrethroids (PYR) are among the most widely used insecticides in households, leading to substantial exposure. Children and adolescents, especially during growth spurts, have a reduced capacity to effectively metabolize these insecticides. The relationship between PYR exposure and asthma in these age groups remains poorly understood, highlighting the need for further research.We used data from the 2007-2014 National Health and Nutrition Examination Survey, which included 1181 children aged 6-11 years and 1258 adolescents aged 12-19 years. The concentration of the PYR metabolite 3-phenoxybenzoic acid (3-PBA) in urine was quantified using solid-phase extraction-high-performance liquid chromatography-heated electrospray ionization tandem mass spectrometry. Asthma was defined based on self-reported doctor diagnoses from the questionnaire. PYR exposure was measured using urine samples collected simultaneously with the questionnaire. We explored the association between PYR exposure and asthma using multiple logistic regression analyses, adjusting for potential confounders.Multiple logistic regression analyses revealed no significant association between PYR exposure and asthma in children and adolescent boys (all P > 0.05). In contrast, PYR exposure was significantly associated with asthma in adolescent girls aged 12-19 years. Specifically, for "ever asthma," the odds ratios (ORs) were 2.49 (95% CI = 1.03-5.97) in the second quartile of PYR exposure and 2.48 (95% CI = 1.04-5.91) in the third quartile, each in comparison to the first quartile. For "current asthma," in comparison to the first quartile, the ORs were 3.99 (95% CI = 1.55-10.26) in the second quartile of PYR exposure, 3.39 (95% CI = 1.32-8.70) in the third quartile, and 2.93 (95% CI = 1.24-6.90) in the fourth quartile.Conclusions:Our study found a significant association between PYR exposure and asthma in adolescent girls, whereas no significant association was observed in children and adolescent boys. These findings suggest potential sex and age differences in susceptibility to PYR exposure. Further research is warranted to confirm these results and elucidate the underlying mechanisms.
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Clostridioides difficile and its endospores possess the characteristics of a foodborne pathogen and have been detected at several stages in the food chain. In the presence of an imbalance in host intestinal ecology, C. difficile can proliferate and cause intestinal infections. Multiple food source factors can substantially alter the host's gut ecosystem, including the consumption of baijiu. However, it remains to be known whether the gut ecological changes induced by the consumption of baijiu increase the risk of C. difficile invasion and infection. In this study, C. difficile cells were exposed to two commercially available baijiu to evaluate the effect of baijiu on C. difficile cells and to verify through a mouse model. The results showed that baijiu effectively inhibited the growth and biofilm production of C. difficile, downregulated the expression levels of tcdA and tcdB virulence genes but upregulated the expression level of spore-producing genes Spo0A, enhanced the spore production, as well as increased C. difficile cell adhesion to Caco-2 cells. The mouse model showed that the intake of baijiu promoted the invasion and infection of C. difficile spores, causing damage to the cecum tissue, accompanied by an increase in the gut lipid carrier protein-2 (Lcn-2) and TcdA toxin protein levels. Simultaneously, cholic acid was elevated, whereas deoxycholic acid was decreased. This study is the first to find a possible link between baijiu intake and C. difficile spore invasion and infection.
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Excessive productions of inflammatory cytokines and free radicals are involved in spinal cord injury (SCI). Fibroblast growth factor 5 (FGF5) is associated with inflammatory response and oxidative damage, and we herein intend to determine its function in SCI. Lentivirus was instilled to overexpress or knockdown FGF5 expression in mice. Compound C or H89 2HCl were used to suppress AMP-activated protein kinase (AMPK) or protein kinase A (PKA), respectively. FGF5 level was significantly decreased during SCI. FGF5 overexpression mitigated, while FGF5 silence further facilitated inflammatory response, oxidative damage and SCI. Mechanically, FGF5 activated AMPK to attenuate SCI in a cAMP/PKA-dependent manner, while inhibiting AMPK or PKA with pharmacological methods significantly abolished the neuroprotective effects of FGF5 against SCI. More importantly, serum FGF5 level was decreased in SCI patients, and elevated serum FGF5 level often indicate better prognosis. Our study identifies FGF5 as an effective therapeutic and prognostic target for SCI.
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Proteínas Quinasas Activadas por AMP , Factor 5 de Crecimiento de Fibroblastos , Estrés Oxidativo , Traumatismos de la Médula Espinal , Animales , Humanos , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Factor 5 de Crecimiento de Fibroblastos/genética , Factor 5 de Crecimiento de Fibroblastos/metabolismo , Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Ratones Noqueados , Masculino , Femenino , Adulto , Persona de Mediana EdadRESUMEN
Hybridization is recognized as a major force in species evolution and biodiversity formation, generally leading to the origin and differentiation of new species. Multiple hybridization events cannot easily be reconstructed, yet they offer the potential to study a number of evolutionary processes. Here, we used nuclear expressed sequence tag-simple sequence repeat and large-scale single nucleotide polymorphism variation data, combined with niche analysis, to investigate the putative independent hybridization events in Notopterygium, a group of perennial herb plants endemic to China. Population genomic analysis indicated that the four studied species are genetically well-delimited and that N. forrestii and N. oviforme have originated by hybridization. According to Approximate Bayesian Computation, the best-fit model involved the formation of N. forrestii from the crossing of N. franchetii and N. incisum, with N. forrestii further backcrossing to N. franchetii to form N. oviforme. The niche analyses indicated that niche divergence [likely triggered by the regional climate changes, particularly the intensification of East Asian winter monsoon, and tectonic movements (affecting both Qinghai-Tibetan Plateau and Qinling Mountains)] may have promoted and maintained the reproductive isolation among hybrid species. N. forrestii shows ecological specialization with respect to their parental species, whereas N. oviforme has completely shifted its niche. These results suggested that the climate and environmental factors together triggered the two-step hybridization of the East Asia herb plants. Our study also emphasizes the power of genome-wide SNPs for investigating suspected cases of hybridization, particularly unravelling old hybridization events.
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Apiaceae , Hibridación Genética , Apiaceae/genética , Teorema de Bayes , Ecosistema , Metagenómica , FilogeniaRESUMEN
Honey-processed Astragalus is a dosage form of Radix Astragalus mixed with honey by a traditional Chinese medicine processing method which improves immune activity. This pharmacological activity of honey-processed Astragalus polysaccharide (HP-APS) might be due to structural changes during the honey roasting process. Previously, we have prepared and characterized HP-APS and preliminarily found its anti-inflammatory effects. However, whether the pharmacodynamic activity of HP-APS induces tumor cell apoptosis and the mechanisms responsible for the immunogenic death (ICD) have not been elucidated. Here, A549, MC38 and B16 cells were used to evaluate the cells viability, apoptosis and cell cycles, respectively. Cellular immunogenic cell death-related molecules calreticulin (CRT), Heat Shock Proteins (HSP)70, major histocompatibility complex I (MHC-I), and co-stimulator molecules CD80/CD86 were determined by flow cytometry. The extracellular ATP release was also detected. B16-OVA and MC38-OVA cells were treated with HP-APS and co-cultivated with OT1 mouse of CD3+T cells for assessment of proliferation, in mice model, and the establishment of C57BL/B6 mouse model bearing B16 cells for assessment of HP-APS the regulation of immune activity in vivo. Our results showed that HP-APS has an inhibitory effect on tumor cell proliferation, which induces tumor cell apoptosis, preventing cells-transforming from G1 phase to S phase in cell cycles. Furthermore, HP-APS could effectively increase the expression of HSP70, CRT, MHC-I, CD86, CD80 and ATP release. T cell proliferation index is significantly improved. CD3 cell proliferation in OT1 mice was significantly increased from the 4th generation to the 5th generation. Moreover, the results have also shown that HP-APS could inhibit tumor growth by increasing immune cell infiltration in the tumor tissues. In the mouse melanoma model with HP-APS treatment, the tumor weight and volume were significantly reduced, and the growth of melanoma was inhibited. CD8+ T is significantly increased. The ratio of CD4+ T and CD8+ T cells numbers are also significantly increased in mouse spleen, but it is less than PD-1 alone treatment separately. Altogether, these findings suggest that HP-APS exerts anti-tumor effects, and that its underlying mechanisms might be associated with the expression of immunogenicity cell death related molecules and the immunomodulatory effects of immune cells.
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Planta del Astrágalo/química , Medicamentos Herbarios Chinos/farmacología , Muerte Celular Inmunogénica/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Células A549 , Animales , Apoptosis/efectos de los fármacos , Astragalus propinquus/química , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Miel/análisis , Humanos , Muerte Celular Inmunogénica/inmunología , Inmunomodulación/efectos de los fármacos , Inmunomodulación/inmunología , Activación de Linfocitos/efectos de los fármacos , Melanoma Experimental , Ratones , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Polisacáridos/química , Polisacáridos/farmacologíaRESUMEN
Anti-vascular endothelial growth factor (VEGF) drugs have revolutionized the treatment of neovascular eye diseases, but responses are incomplete in some patients. Recent evidence shows that integrins are involved in the pathogenesis of neovascular age-related macular degeneration and diabetic retinopathy. JP1, derived from an optimized seven-amino-acid fragment of JWA protein, is a polypeptide specifically targeting integrin αVß3. In this study we evaluated the efficacy of JP1 on laser-induced choroidal neovascularization (CNV) and retinal vascular leakage. CNV mice received a single intravitreal (IVT) injection of JP1 (10, 20, 40 µg) or ranibizumab (RBZ, 10 µg). We showed that JP1 injection dose-dependently inhibited laser-induced CNV; the effect of RBZ was comparable to that of 20 µg JP1; a combined IVT injection of JP1 (20 µg) and RBZ (5 µg) exerted a synergistic effect on CNV. In the 3rd month after streptozotocin injection, diabetic mice receiving IVT injection of JP1 (40 µg) or RBZ (10 µg) once a week for 4 weeks showed significantly suppressed retinal vascular leakage. In both in vivo and in vitro experiments, JP1 counteracted oxidative stress and inflammation via inhibiting ROS/NF-κB signaling in microglial cells, and angiogenesis via modulating MEK1/2-SP1-integrin αVß3 and TRIM25-SP1-MMP2 axes in vascular endothelial cells. In addition, intraperitoneal injection of JP1 (1, 5 or 10 mg) once every other day for 3 times also dose-dependently inhibited CNV. After intraperitoneal injection of FITC-labeled JP1 (FITC-JP1) or FITC in laser-induced CNV mice, the fluorescence intensity in the CNV lesion was markedly increased in FITC-JP1 group, compared with that in FITC group, confirming that JP1 could penetrate the blood-retinal barrier to target CNV lesion. We conclude that JP1 can be used to design novel CNV-targeting therapeutic agents that may replace current invasive intraocular injections.
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Neovascularización Coroidal , Diabetes Mellitus Experimental , Retinopatía Diabética , Animales , Ratones , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Diabetes Mellitus Experimental/tratamiento farmacológico , Retinopatía Diabética/tratamiento farmacológico , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Fluoresceína-5-Isotiocianato/uso terapéutico , Integrina alfaVbeta3/uso terapéutico , Péptidos/uso terapéuticoRESUMEN
BACKGROUND: Astragali Radix (also known as Astragulus) is a traditional medicinal and edible homologous plant for tonifying Qi. Honey-processed Astragalus is a dosage form of Astragali Radix processed with honey, which exhibited better efficacy of tonifying Qi than the raw product. Polysaccharides are their main active components. RESULTS: APS2a and HAPS2a were initially isolated from Astragulus and honey-processed Astragulus. Both of them are highly branched acidic heteropolysaccharides containing É-configuration and ß-configuration glycosidic bonds. The molecular weight and the molecular dimension of HAPS2a decreased and the GalA contained in APS2a was converted to Gal in HAPS2a. The α-configuration galactose residue 1,3,4-α-Galp in the backbone of APS2a was converted to the corresponding ß-configuration galactose residue 1,3,4-ß-Galp in the backbone of HAPS2a and the uronic acid residue T-α-GalpA in the sidechain of APS2a was converted to the corresponding neutral residue T-α-Galp in the side chain of HAPS2a. Bioactivity results showed that HAPS2a had better probiotic effects on Bacteroides ovatus, Bacteroides thetaiotaomicron, Bifidobacterium longum and Lactobacillus rhamnosus strains than APS2a. After degradation, the molecular weights of HAPS2a and APS2a decreased with the changes in their monosaccharide composition. The contents of total short-chain fatty acids (SCFAs) and other organic acids in HAPS2a group were higher than APS2a group. CONCLUSIONS: Two novel high-molecular-weight polysaccharides named APS2a and HAPS2a had different probiotic activities in vitro, which might be due to their structural differences before and after honey processing. Both of them might be possibly used as an immunopotentiator in healthy foods or dietary supplement. © 2023 Society of Chemical Industry.
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Planta del Astrágalo , Microbioma Gastrointestinal , Miel , Humanos , Galactosa , Miel/análisis , Polisacáridos/química , Planta del Astrágalo/químicaRESUMEN
Inflammasome involvement in Parkinson's disease (PD) has been intensively investigated. Absent in melanoma 2 (AIM2) is an essential inflammasome protein known to contribute to the development of several neurological diseases. However, a specific role for AIM2 in PD has not been reported. In this study, we investigated the effect of AIM2 in the N-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced PD model by use of various knockout and bone marrow chimeric mice. The mechanism of action for AIM2 in PD was assessed by RNA-sequencing and in vitro primary microglial transfection. Results were validated in the A30P transgenic mouse model of PD. In the MPTP mouse model, AIM2 activation was found to negatively regulate neuro-inflammation independent of the inflammasome. Microglial AIM2 deficiency exacerbated behavioral and pathological features of both MPTP-induced and transgenic PD mouse models. Mechanistically, AIM2 reduced cyclic GMP-AMP synthase (cGAS)-mediated antiviral-related inflammation by inhibition of AKT-interferon regulatory factor 3 (IRF3) phosphorylation. These results demonstrate microglial AIM2 to inhibit the antiviral-related neuro-inflammation associated with PD and provide for a foundation upon which to identify new therapeutic targets for treatment of the disease.
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Melanoma , Enfermedad de Parkinson , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Antivirales/farmacología , Proteínas de Unión al ADN , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Factor 3 Regulador del Interferón/farmacología , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/metabolismo , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , ARN/metabolismoRESUMEN
New approaches for the engineering of well-defined, pore modality, and multi-chemical functionality nanocomposites are crucial to generate the next generation of functional materials with recoverable and easy preparation properties. Here, a catalyst and heat free polymerization reaction is exploited and fabricated zwitterionic system around magnetic nanoparticles. N-aminoethyl piperazine propane sulfonate (AEPPS) and dopamine (DA) are introduced as the zwitterionic system, which provided abundant zwitterionic groups (NH2, SO3-, N+) and strong adhesion and various oxidation state properties. And that, the zwitterionic engineering will assemble between AEPPS and DA whereby Schiff base formation or Michael type addition. Whereafter, a series of sophisticated array of microscopic, spectroscopic, and structure techniques verify the formation of highly crosslinking internal zwitterionic architectures, well-defined core-shell structure, and better porosity. The zwitterionic structure-function relationships and striking porous structure are explored in a multi-interaction adsorption assay. The adsorption capacity of the magnetic nanocomposites was 1065.8 mg/g. And that, the system exhibited with hydrophilic-hydrophobic activity towards glycoprotein and better performance to bioactive protein (Ig-G) isolation form human whole blood sample. The synergistic enhancement interaction in hydrophilic target enrichment, easy preparation, and soft substrate properties of the AEPPS-DA zwitterionic materials make them intriguing candidates for sustainable biomedical loading and chromatographic separation.
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Nanocompuestos , Glicoproteínas , Humanos , Fenómenos Magnéticos , Magnetismo , Nanocompuestos/química , PolimerizacionRESUMEN
Probiotics have the potential to be used in the prevention of Clostridioides difficile infection (CDI). In this study, selenium (Se)-enriched Bifidobacterium breve YH68-Se was obtained under optimal culture conditions with single-factor and response surface optimization. The overall environmental resistance of YH68-Se was superior to that of the parental strain YH68, mainly reflected in the substantial improvement of antioxidant activity and gastrointestinal tolerance. YH68-Se dramatically inhibited C. difficile growth, spore, biofilm, toxin production, and virulence gene expression, rapidly disrupted C. difficile cell membrane permeability and integrity, and altered the membrane proton motive force (PMF), induced a large outflow of intracellular substances and eventually caused bacterial death. The main factor inducing this process originated from the lactic acid (LD) in YH68-Se. In addition, the LD production of YH68 increased with increasing selenite concentration and was accompanied by enhanced activities of thioredoxin reductase (TrxR), glutathione peroxidase (GSH-Px), and increased concentration of autoinducer-2 (AI-2), which may be the crucial factors contributing to the outstanding probiotic properties of YH68-Se and their potent antagonism of C. difficile. KEY POINTS: ⢠Compared with the parental strain B. breve YH68, the environmental resistance of YH68-Se was improved. ⢠YH68-Se was able to produce more lactic acid, which suppressed the important physiological activities of C. difficile and rapidly disrupted their cell membrane structures. ⢠Sodium selenite in the suitable concentration range gradually increases the yield of lactic acid and phenylacetic acid, increased the concentration of autoinducer-2, and enhanced the activities of antioxidant enzymes TrxR and GSH-Px in YH68.
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Bifidobacterium breve , Clostridioides difficile , Selenio , Antioxidantes , Bifidobacterium breve/metabolismo , Clostridioides , Glutatión Peroxidasa/metabolismo , Ácido Láctico , Selenio/metabolismoRESUMEN
A novel peptide containing antimicrobial sequence and gelatinase cleavage sites was designed for Staphylococcus aureus detection. Since Staphylococcus aureus could secrete gelatinase, the fluorescein labeled peptide GKRWWKWWRRPLGVRGC could be recognized and cleaved. The obtained products were able to be analyzed by capillary electrophoresis with fluorescence detection. To explore the effect of Staphylococcus aureus concentration on enzyme digestion ability of peptide, Staphylococcus aureus with different concentrations were incubated with the peptide. Results indicated that capillary electrophoretic method was efficient for determining Staphylococcus aureus content. Compared with traditional approaches for Staphylococcus aureus detection, capillary electrophoresis possessed higher efficiency, enhanced sensitivity, and low sample consumption. Moreover, the proposed peptide also presented desirable antimicrobial activity. It suggested that the novel antimicrobial peptide used in this research opens a new path of detecting Staphylococcus aureus by capillary electrophoretic method.
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Péptidos Antimicrobianos , Staphylococcus aureus , Secuencia de Aminoácidos , Electroforesis Capilar , Fluoresceína , Gelatinasas , Staphylococcus aureus/aislamiento & purificaciónRESUMEN
Batch low-pH hold is a common processing step to inactivate enveloped viruses for biologics derived from mammalian sources. Increased interest in the transition of biopharmaceutical manufacturing from batch to continuous operation resulted in numerous attempts to adapt batch low-pH hold to continuous processing. However, control challenges with operating this system have not been directly addressed. This article describes a low-cost, column-based continuous viral inactivation system constructed with off-the-shelf components. Model-based, reaction-invariant pH controller is implemented to account for the nonlinearities with Bayesian estimation addressing variations in the operation. The residence time distribution is modeled as a plug flow reactor with axial dispersion in series with a continuously stirred tank reactor, and is periodically estimated during operation through inverse tracer experiments. The estimated residence time distribution quantifies the minimum residence time, which is used to adjust feed flow rates. Controller validation experiments demonstrate that pH and minimum residence time setpoint tracking and disturbance rejection are achieved with fast and accurate response and no instability. Viral inactivation testing demonstrates tight control of logarithmic reduction values over extended operation. This study provides tools for the design and operation of continuous viral inactivation systems in service of increasing productivity, improving product quality, and enhancing patient safety.
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Productos Biológicos , Modelos Químicos , Inactivación de Virus , Humanos , Concentración de Iones de HidrógenoRESUMEN
The use of adenosine monophosphate challenge and basal cortisol as short-term surrogate end points of airway-systemic effects of inhaled corticosteroids in asthma is not suitable to properly determine the clinically relevant long-term therapeutic index.
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Antiasmáticos , Asma , Administración por Inhalación , Corticoesteroides/efectos adversos , Antiasmáticos/efectos adversos , Asma/tratamiento farmacológico , Humanos , Hidrocortisona/uso terapéuticoRESUMEN
BACKGROUND: Prodrugs are medications or compounds that, after administration, can be converted into pharmacologically active drugs through metabolism. Unlike conventional drugs, prodrugs have reduced adverse or unintended effects, which could become critical limitations in treatments such as chemotherapy. Previously through computer-aided drug design and chemical synthesis, we have obtained and examined a prodrug N-benzyloxycarbonyl-Ala-Asn-Doxorubicin (CBZ-AAN-DOX). CBZ-AAN-DOX is essentially Doxorubicin that is chemically-modified with tripeptides to target Legumain, a highly expressed protein in cancer cells and is involved in tumor metastasis and tumor microvessel formation. The difficulty to test the safety and efficacy of the prodrug (including the pharmacodynamic parameters of CBZ-AAN-DOX on metastasis and invasion of tumors, as well as cardiac and vascular toxicity) primarily comes from the lack of appropriate experimental models. METHODS: Human cervical cancer cell lines CaSki under hypoxic conditions were used to evaluate the cell viability by CCK-8 assay after the prodrug treatment. Western blotting method was performed for Legumain protein determination in the cell culture. Wound healing and transwell invasion assays were performed to determine the effects of the prodrug on tumor metastasis and invasion, respectively. Zebrafish models were constructed for toxicity and angiogenesis visual analysis after in vivo treatment with the prodrug. RESULTS: The CCK-8 results showed that CBZ-AAN-DOX exhibits an IC50 of 28.7 µM in 48 h on CaSki cells that had a lower cell inhibition rate than DOX 80.3 µM for 24 h. Legumain expression was significantly increased in a time-dependent manner in 48 h under hypoxia conditions. The results also showed that 13.9 µM of the prodrug significantly inhibited the migration and invasion of cells and the effects were significantly stronger than that of 41.8 µM of DOX under hypoxia conditions after 48 h. The effects of 160 µM of the prodrug on the survival rate of zebrafish after 72 h and heart-toxicity showed no obvious abnormalities. Cell metastasis and angiogenesis were also inhibited in tumor-bearing zebrafish model. CONCLUSION: The findings in this study demonstrated that CBZ-AAN-DOX is a promising chemotherapy candidate with low toxicity and high efficiency for cervical cancer. Remarkably, the hypoxic culture model together with the zebrafish model serve as a good system for the evaluation of the toxicity, targeting and impact of the prodrug on tumor invasion and metastasis.
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Antibióticos Antineoplásicos/farmacología , Cisteína Endopeptidasas/genética , Doxorrubicina/análogos & derivados , Regulación Neoplásica de la Expresión Génica , Neovascularización Patológica/prevención & control , Oligopéptidos/farmacología , Profármacos/farmacología , Neoplasias del Cuello Uterino/tratamiento farmacológico , Animales , Antibióticos Antineoplásicos/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular , Cisteína Endopeptidasas/metabolismo , Modelos Animales de Enfermedad , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Femenino , Humanos , Hipoxia/diagnóstico , Hipoxia/tratamiento farmacológico , Hipoxia/genética , Hipoxia/mortalidad , Neovascularización Patológica/diagnóstico , Neovascularización Patológica/genética , Neovascularización Patológica/mortalidad , Oligopéptidos/metabolismo , Profármacos/metabolismo , Análisis de Supervivencia , Neoplasias del Cuello Uterino/diagnóstico , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/mortalidad , Ensayos Antitumor por Modelo de Xenoinjerto , Pez CebraRESUMEN
Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o- (16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 104 macrophages/cm2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing.
RESUMEN
INTRODUCTION: Polygoni Multiflori Caulis (PMC) has been used as a traditional Chinese medicine for a long time in China. However, hepatotoxic events of PMC have been reported in recent years, but the potential toxic compounds have remained unclear. Dianthrones as the secondary plant metabolites were revealed to potential hepatotoxicity in a previous study. However, no reports focused on dianthrones in PMC. OBJECTIVE: In the quest for exploring potential hepatotoxic compounds in PMC, the aim of this work was to undertake a comprehensive characterisation of dianthrones in PMC. METHODS: A simple and effective macroporous absorbent resin column chromatography method was established in this study to enrich the minor dianthrones from PMC extracts. Exploration and characterisation of dianthrones in PMC was conducted by an ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS/MS) method and information dependent acquisition (IDA) mode. The aglycones of dianthrone glycosides were further verified by acid hydrolysis method. RESULTS: Seventy-two dianthrone glycosides and their five aglycones were discovered and tentatively characterised in PMC for the first time, of which 29 dianthrones were identified as potential new compounds. Dianthrone glycosides could be classified into three types according to their aglycone structures, and their fragmentation pathway rules and diagnosed ions were also summarised comprehensively. CONCLUSION: This was the first comprehensive investigation on dianthrones in PMC. The result would help to fully understand the phytochemical constituents and toxic components in PMC, and highlight the need for further toxicological investigations of the dianthrones in PMC due to their potential hepatotoxicity correlation.