RESUMEN
D-Amino acid oxidase (DAAO) selectively catalyzes the oxidative deamination of D-amino acids, making it one of the most promising routes for synthesizing optically pure L-amino acids, including L-phosphinothricin ( L-PPT), a chiral herbicide with significant market potential. However, the native DAAOs that have been reported have low activity against unnatural acid substrate D-PPT. Herein, we designed and screened a DAAO from Rhodotorula taiwanensis (RtwDAAO), and improved its catalytic potential toward D-PPT through protein engineering. A semirational design approach was employed to create a mutation library based on the tunnel-pocket engineering. After three rounds of iterative saturation mutagenesis, the optimal variant M3rd -SHVG was obtained, exhibiting a >2000-fold increase in relative activity. The kinetic parameters showed that M3rd -SHVG improved the substrate binding affinity and turnover number. This is the optimal parameter reported so far. Further, molecular dynamics simulation revealed that the M3rd -SHVG reshapes the tunnel-pocket and corrects the direction of enzyme-substrate binding, allowing efficiently catalyze unnatural substrates. Our strategy demonstrates that the redesign of tunnel-pockets is effective in improving the activity and kinetic efficiency of DAAO, which provides a valuable reference for enzymatic catalysis. With the M3rd -SHVG as biocatalyst, 500 mM D, L-PPT was completely converted and the yield reached 98%. The results laid the foundation for further industrial production.
Asunto(s)
Aminoácidos , Ingeniería de Proteínas , Especificidad por Sustrato , Aminoácidos/metabolismo , Unión Proteica , Oxidorreductasas/metabolismo , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/química , D-Aminoácido Oxidasa/metabolismo , CinéticaRESUMEN
BACKGROUND/AIM: Rheumatoid arthritis (RA) is an inflammatory autoimmune disease, and management of it is still a challenge. The present investigation assessed the potential preventive effect of phlorizin on rats with RA. MATERIALS AND METHODS: A total of 40 healthy Wistar rats were used for this study. Bovine type II collagen and Freund's incomplete adjuvant (1:1 and 1 mg/ml) were administered on days 1 and 8 of the protocol to induce RA in rats; treatment with phlorizin at 60 or 120 mg/kg was started after the 4th week of the protocol, and its effect on inflammation, level of inflammatory cytokines, and expression of proteins were estimated in RA rats. Moreover, an in vitro study was performed on fibroblast-like synoviocytes (FLSs), and the effects of phlorizin on proliferation, apoptosis, and expression of the mechanistic target of rapamycin kinase pathway protein after stimulating these cells with tumor necrosis factor α (TNF-α) were estimated. RESULTS: The data obtained from the study indicate that phlorizin has the potential to mitigate inflammation and enhance weight management in rats with RA induced by bovine type II collagen (CII). The level of inflammatory cytokines in the serum and the expression of protein kinase B (AKT), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), and mechanistic target of rapamycin kinase (mTOR) proteins in the joint tissue were reduced in phlorizin-treated rats with RA. In this investigation, phlorizin was shown to reverse the histological abnormalities in the joint tissue of rats with RA. The in-vitro study showed that phlorizin reduced proliferation and had no apoptotic effect on TNF-α-stimulated FLSs. Expression of AKT, PI3K, and mTOR proteins was also down-regulated in phlorizin-treated TNF-α-stimulated FLSs. CONCLUSION: Phlorizin protects against inflammation and reduces injury to synovial tissues in RA by modulating the AKT/PI3K/mTOR pathway.
Asunto(s)
Artritis Reumatoide , Hiperplasia , Inflamación , Florizina , Transducción de Señal , Sinoviocitos , Serina-Treonina Quinasas TOR , Animales , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Serina-Treonina Quinasas TOR/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Florizina/farmacología , Inflamación/patología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Sinoviocitos/efectos de los fármacos , Sinoviocitos/metabolismo , Sinoviocitos/patología , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Modelos Animales de Enfermedad , Citocinas/metabolismo , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Masculino , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Artritis Experimental/metabolismo , Ratas Wistar , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease and management of it still a challenge. Given report evaluates protective effect of phlorizin on RA and also postulates the molecular mechanism of its action. Bovine type II collagen (CIA) and Freund's incomplete adjuvant (1:1 and 1 mg/ml) was administered on 1st and 8th day of protocol to induce RA in rats and treatment with phlorizin 60 and 120 mg/kg was started after 4th week of protocol. Level of inflammatory cytokines and expression of proteins were estimated in phlorizin treated RA rats. Moreover in-vitro study was performed on Fibroblast-like synoviocytes (FLSs) and effect of phlorizin was estimated on proliferation, apoptosis and expression of mTOR pathway protein after stimulating these cell lines with Tumour Necrosis Factor alpha (TNF-α). Data of study suggest that phlorizin reduces inflammation and improves weight in CIA induced RA rats. Level of inflammatory cytokines in the serum and expression of Akt/PI3K/mTOR proteins in the join tissue was reduced in phlorizin treated RA rats. Phlorizin also reported to reverse the histopathological changes in the joint tissue of RA rats. In-vitro study supports that phlorizin reduces proliferation and no apoptotic effect on TNF-α stimulated FLSs. Expression of Akt/PI3K/mTOR proteins also downregulated in phlorizin treated TNF-α stimulated FLSs. In conclusion, phlorizin protects inflammation and reduces injury to the synovial tissues in RA, as it reduces autophagy by regulating Akt/PI3K/mTOR pathway.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Hiperplasia , Florizina , Sinoviocitos , Serina-Treonina Quinasas TOR , Animales , Humanos , Masculino , Ratas , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Apoptosis/efectos de los fármacos , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/patología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/metabolismo , Hiperplasia/tratamiento farmacológico , Florizina/farmacología , Florizina/uso terapéutico , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/patología , Sinoviocitos/efectos de los fármacos , Sinoviocitos/patología , Serina-Treonina Quinasas TOR/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The d-amino acid oxidase (DAAO) is pivotal in obtaining optically pure l-glufosinate (l-PPT) by converting d-glufosinate (d-PPT) to its deamination product. We screened and designed a Rasamsonia emersonii DAAO (ReDAAO), making it more suitable for oxidizing d-PPT. Using Caver 3.0, we delineated three substrate binding pockets and, via alanine scanning, identified nearby key residues. Pinpointing key residues influencing activity, we applied virtual saturation mutagenesis (VSM), and experimentally validated mutants which reduced substrate binding energy. Analysis of positive mutants revealed elongated side-chain prevalence in substrate binding pocket periphery. Although computer-aided approaches can rapidly identify advantageous mutants and guide further design, the mutations obtained in the first round may not be suitable for combination with other advantageous mutations. Therefore, each round of combination requires reasonable iteration. Employing VSM-assisted screening multiple times and after four rounds of combining mutations, we ultimately obtained a mutant, N53V/F57Q/V94R/V242R, resulting in a mutant with a 5097% increase in enzyme activity compared to the wild type. It provides valuable insights into the structural determinants of enzyme activity and introduces a novel rational design procedure.
Asunto(s)
D-Aminoácido Oxidasa , Ingeniería de Proteínas , D-Aminoácido Oxidasa/genética , D-Aminoácido Oxidasa/metabolismo , D-Aminoácido Oxidasa/química , Ingeniería de Proteínas/métodos , Especificidad por Sustrato , Mutagénesis , Mutagénesis Sitio-Dirigida/métodos , Aminobutiratos/metabolismo , Modelos Moleculares , Mutación , Sitios de UniónRESUMEN
OBJECTIVES: To compare the clinical effect between two acupoint regimens of moxibustion on knee osteoarthritis (KOA), and observe the influences on the serum content of interleukin 1α (IL-1α), interleukin-17A (IL-17A), tumor necrosis factor α (TNF-α), bone gla protein (BGP) and osteoprotegerin (OPG). METHODS: KOA patients were randomly divided into an observation group (40 cases, 2 cases dropped off) and a control group (40 cases, 3 cases dropped off). In the observation group, moxibustion was applied to Xiyan (EX-LE5), Dubi (ST35), Zusanli (ST36), Dazhu (BL11), Xuanzhong (GB39) and Yongquan (KI1) on the affected side. In the control group, EX-LE5, ST35 and ST36 were selected on the affected side. One session of treatment took 30 min in each group, delivered 3 times a week and the duration of treatment was 4 weeks. The scores of Western Ontario and McMaster University (WOMAC) and visual analogue scale (VAS) were observed and the serum content of IL-1α, IL-17A, TNF-α, BGP and OPG of the two groups were measured before and after treatment. RESULTS: Compared with those before treatment, the WOMAC score, VAS score and the serum content of IL-1α, IL-17A and TNF-α were decreased (P<0.05), and the content of BGP and OPG were increased (P<0.05) after treatment. Compared with the control group, the WOMAC score, VAS score and the serum content of IL-1α and TNF-α in the observation group were lower (P<0.05), and the content of BGP and OPG were higher (P<0.05). The total effective rate of the observation group was 89.5% (34/38), and that of the control group was 83.8% (31/37), with no statistically significant difference. CONCLUSIONS: Moxibustion therapy of "nourishing the kidney and benefiting the marrow" can relieve joint pain, improve joint function, reduce the level of inflammatory factors and ameliorate bone metabolic indicators. The effect of the acupoint regimen in this moxibustion therapy is better than that of the local acupoint selection.
Asunto(s)
Moxibustión , Osteoartritis de la Rodilla , Humanos , Osteoartritis de la Rodilla/terapia , Interleucina-17 , Médula Ósea , Factor de Necrosis Tumoral alfa , Resultado del Tratamiento , Puntos de Acupuntura , RiñónRESUMEN
BACKGROUND: Biocatalytic production of L-phosphinothricin (L-PPT) is currently the most promising method. In this work, we use an Escherichia coli strain coexpressing of D-amino acid oxidase and catalase (E. coli DAAO-CAT) to oxidation biocatalytic D-PPT to PPO, then use the second E. coli strain coexpressing glutamate dehydrogenase and formate dehydrogenase (E. coli GluDH-FDH) to reduce biocatalytic PPO to L-PPT. MAIN METHODS AND MAJOR RESULTS: We compared the effects of different concentrations of IPTG or lactose on protein expression and enzyme activity in 5 L fermenter. The best induction conditions for E. coli DAAO-CAT were 0.05 mM IPTG, induction for 18 h at 28°C. The specific enzyme activities of DAAO and CAT were 153.20 U g-1 and 896.23 U g-1 , respectively. The optimal induction conditions for E. coli GluDH-FDH were 0.2 mM IPTG, induction for 19 h at 28°C. The specific enzyme activities of GluDH and FDH were 41.72 U g-1 and 109.70 U g-1 , respectively. The 200 mM D-PPT was biocatalyzed by E. coli DAAO-CAT for 4 h with space-time yield of 9.0 g·L-1 ·h-1 and conversion rate of over 99.0%. Then 220 mM PPO was converted to L-PPT by E. coli GluDH-FDH for 3 h with space-time yield of 14.5 g·L-1 ·h-1 and conversion rate of over 99.0%. To our knowledge, this is the most efficient biocatalytic reaction for L-PPT production. CONCLUSIONS AND IMPLICATIONS: We found that IPTG has advantages compared with lactose in the enzyme activity and biomass of E. coli DAAO-CAT and E. coli GluDH-FDH, and IPTG is more environmentally friendly. Our data implicated that IPTG can replace lactose in terms of economic feasibility and effectiveness for scaled-up industrial fermentations.
Asunto(s)
Escherichia coli , Lactosa , Isopropil Tiogalactósido/metabolismo , Isopropil Tiogalactósido/farmacología , Escherichia coli/metabolismo , Lactosa/metabolismo , Glutamato Deshidrogenasa/metabolismoRESUMEN
The d-amino acid oxidase (DAAO) from Rhodotorula taiwanensis has proven to have great potential for applications due to its excellent catalytic kinetic parameters. However, its poor thermal stability has limited its performance in biocatalysis. Herein, starting from the variant SHVG of RtwDAAO, this study employed a comprehensive computational design approach for protein stability engineering, resulting in positive substitutions at specific sites (A43S, T45M, C234L, E195Y). The generated variant combination, SHVG/SMLY, exhibited a significant synergistic effect, leading to an extension of the half-life and Tmapp. The ancestral sequence reconstruction revealed the conservation of the variant sites. The association of the variant sites with the highly stable ancestral enzyme was further explored. After determining the contribution of the variant sites to thermal stability, it was applied to other homologous sequences and validated. Molecular dynamics simulations indicated that the increased hydrophobicity of the variant SHVG/SMLY was a key factor for the increased stability, with strengthened intersubunit interactions playing an important role. In addition, the physical properties of the amino acids themselves were identified as crucial factors for thermal stability generality in homologous enzymes, which is important for the rapid acquisition of a series of stable enzymes.
Asunto(s)
Aminoácidos , Ingeniería de Proteínas , Simulación de Dinámica Molecular , Hidrolasas , Estabilidad de Enzimas , CinéticaRESUMEN
BACKGROUND: National and international experts have been attempting to find diagnostic tools for the early identification of symptoms to facilitate early identification and intervention of the disease. OBJECTIVE: Detection of urine Alzheimer-associated neuronal thread protein (AD7c-NTP) and serum 25-hydroxyvitamin D (25(OH)D) in the diagnosis of Alzheimer's disease (AD). METHODS: Subjects aged >50 years who underwent a physical examination at the Taihu Sanatorium of Jiangsu Province, had no clinical evidence of AD-related issues, and had normal Mini-Mental State Exam and Montreal Cognitive Assessment scores were enrolled in the present study. There were 35 males and 15 females, who were aged 51-91 years. Urine AD7c-NTP levels and serum 25(OH)D concentrations were measured. RESULTS: The Pearson correlation analysis revealed that the urine AD7c-NTP levels in these subjects were negatively correlated with the serum 25(OH)D concentrations (râ=â-0.460, pâ<â0.001). CONCLUSION: Combined with previous studies, it was considered that cognitive function might be the only link for the correlation between AD7c-NTP and 25(OH)D. This finding might provide a starting point to investigate the potential value of the interaction between urine AD7c-NTP and serum 25(OH)D in chronic diseases. Further large-scale studies are needed to validate the results of the present study.
Asunto(s)
Enfermedad de Alzheimer , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/orina , Cognición , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Proteínas del Tejido Nervioso , Vitamina D , VitaminasRESUMEN
D-amino acid oxidase (DAAO) is widely used in the industrial preparation of L-amino acids, and cultivating Escherichia coli (E. coli) expressing DAAO for the biosynthesis of L-phosphinothricin (L-PPT) is very attractive. At present, the biomass production of DAAO by fermentation is still limited in large-scale industrial applications because the expression of DAAO during the fermentation process inhibits the growth of host cells, which limits higher cell density. In this study, the factors that inhibit the growth of bacterial cells during a 5-L fed-batch fermentation process were explored, and the fermentation process was optimized by co-expressing catalase (CAT), by balancing the biomass and the enzyme activity, and by adding exogenous D-alanine (D-Ala) to relieve the limitation of DAAO on the cells and optimize fermentation. Under optimal conditions, the DO-STAT feeding mode with DO controlled at 30% ± 5% and the addition of 27.5 g/L lactose mixed with 2 g/L D-Ala during induction at 28 °C resulted in the production of 26.03 g dry cell weight (DCW)/L biomass and 390.0 U/g DCW specific activity of DAAO; an increase of 78% and 84%, respectively, compared with the initial fermentation conditions. The fermentation strategy was successfully scale-up to a 5000-L fermenter.
Asunto(s)
Biomasa , D-Aminoácido Oxidasa/biosíntesis , Escherichia coli/crecimiento & desarrollo , Expresión Génica , D-Aminoácido Oxidasa/genética , Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Osteosarcoma is the most common type of malignant bone tumor, often affecting adolescents and children. MicroRNAs (miRNAs) are a group of small, non-protein coding, endogenous RNAs that play critical roles in osteosarcoma tumorigenesis. In our study, we demonstrated that miR-448 expression was downregulated in osteosarcoma tissues and cell lines. Overexpression of miR-448 suppressed osteosarcoma cell proliferation, colony formation and migration. Moreover, we found that EPHA7 was a direct target gene of miR-448 in osteosarcoma cells. We further demonstrated that the EPHA7 expression level was upregulated in osteosarcoma tissues. Interestingly, the expression level of EPHA7 was inversely correlated with the expression level of miR-448 in osteosarcoma tissues. In addition, elevated expression of miR-448 suppressed osteosarcoma cell proliferation and invasion through targeting EPHA7. Taken together, these findings suggest that miR-448 functioned as a tumor suppressor gene in the development of osteosarcoma through targeting EPHA7.
Asunto(s)
Neoplasias Óseas/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Osteosarcoma/genética , Interferencia de ARN , Receptor EphA7/genética , Emparejamiento Base , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Humanos , Osteosarcoma/patología , Ensayo de Tumor de Célula MadreRESUMEN
A pilot-scale bio-filter was constructed for the removal of high concentrations of iron (TFe 9.0-12.0 mg·L-1, Fe(â ¡) 6.5-8.0 mg·L-1), manganese (1.9-2.1 mg·L-1), and ammonia nitrogen (1.4-1.7 mg·L-1) simultaneously from low temperature (5-6â) groundwater in a plant. The results showed that iron was removed at the beginning of the bio-filter start-up, and manganese and ammonia nitrogen were removed on day 72 and day 75, respectively. The start-up period was influenced by the culture temperature and the raw water quality. For higher filtration rates, the removal of manganese was lower. When the filtration rate was more than 1.0 m·h-1, the maximum removal of manganese was about 3.0 mg·L-1. Manganese was the limiting factor for the increase of filtration rate, and the maximum filtration rate of the single bio-filter was 4.5 m·h-1. When the filtration rate was less than 6.0 m·h-1, the removal of ammonia nitrogen was about 1.5 mg·L-1, which was not affected by the filtration rate. Dissolved oxygen (DO) deficiency led to failure with the removal of more ammonia nitrogen. The required thickness of the bio-filter required for purification increased as the concentration of manganese and ammonia nitrogen increased when DO was sufficient. The removed iron, manganese, and ammonia nitrogen move to the depth of the filter layer, and there will be "manganese dissolution" when the filtration rate is increased. Iron and ammonia nitrogen in the filter layer can be oxidized and removed simultaneously. Manganese is oxidized and removed after the iron and ammonia nitrogen. The effective oxidation and removal section of manganese, iron, and ammonia nitrogen are obviously graded.
Asunto(s)
Agua Subterránea/química , Hierro/aislamiento & purificación , Manganeso/aislamiento & purificación , Nitrógeno/aislamiento & purificación , Contaminantes Químicos del Agua/aislamiento & purificación , Purificación del Agua , Amoníaco/aislamiento & purificación , Filtración , TemperaturaRESUMEN
We explored the desired features of medication applications for patients with chronic disease and their caregivers with a questionnaire survey, 50 from patients and 50 from their caregivers. Although the majority of people (75%) are willing to use medication apps, the actual usage rate is quite low (11%). Worrying about privacy of personal information seems to be the main reason of not using applications. The overall score desired for use was 3.29 ± 1.02 (out of 5). Searching medications and diseases and assistance with making doctors' appointments are the most wanted categories. Online shopping for drugs and delivery were the least desired items. The main concerns for people who do not want certain features include: they are not useful, worrying about buying counterfeit drugs and reliability of content. Compared with patients, caregivers seems to be more concerned on nutrition tips for chronic illness, fall detection, and privacy protection (P < 0.05 for all).