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1دورية أكاديمية
المساهمون: University of Warsaw, Faculty of Chemistry, Warsaw, Poland, Medical University of Warsaw, Faculty of Pharmacy, Department of Laboratory Medicine, Warsaw, Poland
مصطلحات موضوعية: Lactate dehydrogenase, Ferrozine, Nitrotetrazolium Blue, Flow analysis, Multicommutation
الوصف: The routine method for LDH (Lactate dehydrogenase) activity determination is to monitor the increase of NADH concentration at 340 nm. There are some inconvenience in taking measurements in the near-UV region, especially in the case of serum samples analysis. In this work, two modifications of the routine LDH activity assay based on the use of reducing properties of NADH have been compared. Both methods involved the reduction of compounds that can be easily determined by well-known methods, ferric ion (with ferrozine) and nitrotetrazolium blue (NBT). A fully-mechanized Multicommutated Flow Analysis-Paired Emitter Detector Diode (MCFA-PEDD) system based on solenoid devices was developed and applied for both methods. The linear ranges obtained for Fe ferrozine and NBT methods are 6.0-200.0 U L -1 and 10.0-250.0 U L -1 with estimated detection limits at 0.2 U L -1 and 4.5 U L -1 , respectively. The low LOQ values enabled 10-fold sample dilutions, which is advantageous for samples with limited available volume. The Fe-ferrozine method is more selective for LDH activity in the presence of glucose, ascorbic acid, albumin, bilirubin, copper and calcium ions than NBT method. To confirm the analytical usefulness of the proposed flow system, the analysis of real human serum samples was carried out. The statistic tests showed satisfactory correlation between the results obtained for both developed methods and those received using the reference method. ; Polish National Science Centre
وصف الملف: application/pdf
العلاقة: PRELUDIUM21 no. 2022/45/N/NZ5/00953; Głowacka J., Wiśniewska A., Koncki R., Strzelak K., Photometric flow system for the determination of serum lactate dehydrogenase activity. Talanta (2023) 124817 (Zdeponowany zbiór danych jest materiałem źródłowym dla powiązanej publikacji.) doi: https://doi.org/10.1016/j.talanta.2023.124817Test; https://depot.ceon.pl/handle/123456789/22638Test
الإتاحة: https://doi.org/10.1016/j.talanta.2023.124817Test
https://depot.ceon.pl/handle/123456789/22638Test -
2تقرير
المساهمون: Clark, D
المصدر: Other Information: PBD: [1990]
وصف الملف: Medium: ED; Size: 5 p.
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3تقرير
المساهمون: Clark, D
وصف الملف: Medium: ED; Size: Pages: (5 p)
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4تقرير
المؤلفون: Liu, Min, Huo, Meitong, Liu, Changshui, Guo, Likun, Ding, Yamei, Ma, Qingjun, Qi, Qingsheng, Xian, Mo, Zhao, Guang
مصطلحات موضوعية: lysine acetylation, lactate dehydrogenase A, enzyme activity, metabolic engineering, lactate synthesis, Biotechnology & Applied Microbiology, Science & Technology - Other Topics, Multidisciplinary Sciences
الوصف: As an evolutionarily conserved posttranslational modification, protein lysine acetylation plays important roles in many physiological and metabolic processes. However, there are few reports about the applications of lysine acetylation in metabolic regulations. Lactate is a main byproduct in microbial fermentation, and itself also an important bulk chemical with considerable commercial values in many fields. Lactate dehydrogenase (LdhA) is the key enzyme catalyzing lactate synthesis from pyruvate. Here, we reported that Escherichia coli LdhA can be acetylated and the acetylated lysine sites were identified by mass spectrometry. The effects and regulatory mechanisms of acetylated sites on LdhA activity were characterized. Finally, lysine acetylation was successfully used to regulate the lactate synthesis. LdhA (K9R) mutant overexpressed strain improved the lactate titer and glucose conversion efficiency by 1.74 folds than that of wild-type LdhA overexpressed strain. LdhA (K154Q-K248Q) mutant can inhibit lactate accumulation and improve 3HP production. Our study established a paradigm for lysine acetylation in lactate synthesis regulation and suggested that lysine acetylation may be a promising strategy to improve the target production and conversion efficiency in microbial synthesis. The application of lysine acetylation in regulating lactate synthesis also provides a reference for the treatment of lactate-related diseases.
العلاقة: FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY; http://ir.qdio.ac.cn/handle/337002/180867Test; http://ir.qdio.ac.cn/handle/337002/180868Test
الإتاحة: https://doi.org/10.3389/fbioe.2022.966062Test
http://ir.qdio.ac.cn/handle/337002/180867Test
http://ir.qdio.ac.cn/handle/337002/180868Test -
5تقرير
المؤلفون: Maervoet, Veerle, De Maeseneire, Sofie, Avci, Fatma, Beauprez, Joeri, Soetaert, Wim, De Mey, Marjan
مصطلحات موضوعية: 3-propanediol, Glycerol, Citrobacter werkmanii DSM17579, NADH, Lactate dehydrogenase, Ethanol dehydrogenase, Multiple knock-out mutant, Rational engineering
الوقت: 1
الوصف: Background Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii ∆ dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. Results By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA , and ethanol dehydrogenase coded by adhE ) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii ∆ dhaD resulted in the strain C. werkmanii ∆ dhaD ∆ ldhA ∆ adhE ::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. Conclusions In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.
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6تقرير
المؤلفون: de Lima, Pollyne, Mulder, Kelly, Melo, Nadiele, Carvalho, Lucas, Menino, Gisele, Mulinari, Eduardo, de Castro, Virgilio, dos Reis, Thaila, Goldman, Gustavo, Magalhães, Beatriz, Parachin, Nádia
مصطلحات موضوعية: l-Lactic acid, Pichia (Komagataella) pastoris, Lactate transporter, Oxygen limited fermentation, Lactate dehydrogenase
الوصف: Background Crude glycerol is the main byproduct of the biodiesel industry. Although it can have different applications, its purification is costly. Therefore, in this study a biotechnological route has been proposed for further utilization of crude glycerol in the fermentative production of lactic acid. This acid is largely utilized in food, pharmaceutical, textile, and chemical industries, making it the hydroxycarboxylic acid with the highest market potential worldwide. Currently, industrial production of lactic acid is done mainly using sugar as the substrate. Thus here, for the first time, Pichia pastoris has been engineered for heterologous l -lactic acid production using glycerol as a single carbon source. For that, the Bos taurus lactate dehydrogenase gene was introduced into P. pastoris . Moreover, a heterologous and a novel homologous lactate transporter have been evaluated for l -lactic acid production. Results Batch fermentation of the P. pastoris X-33 strain producing LDHb allowed for lactic acid production in this yeast. Although P. pastoris is known for its respiratory metabolism, batch fermentations were performed with different oxygenation levels, indicating that lower oxygen availability increased lactic acid production by 20 %, pushing the yeast towards a fermentative metabolism. Furthermore, a newly putative lactate transporter from P. pastoris named PAS has been identified by search similarity with the lactate transporter from Saccharomyces cerevisiae Jen1p. Both heterologous and homologous transporters, Jen1p and PAS, were evaluated in one strain already containing LDH activity. Fed-batch experiments of P. pastoris strains carrying the lactate transporter were performed with the batch phase at aerobic conditions followed by an aerobic oxygen-limited phase where production of lactic acid was favored. The results showed that the strain containing PAS presented the highest lactic acid titer, reaching a yield of approximately 0.7 g/g. Conclusions We showed that P. pastoris has a great ...
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7تقرير
المؤلفون: Kakugawa, Tomoyuki, Sakamoto, Noriho, Sato, Shuntaro, Yura, Hirokazu, Harada, Tatsuhiko, Nakashima, Shota, Hara, Atsuko, Oda, Keishi, Ishimoto, Hiroshi, Yatera, Kazuhiro, Ishimatsu, Yuji, Obase, Yasushi, Kohno, Shigeru, Mukae, Hiroshi
مصطلحات موضوعية: Bronchoalveolar lavage, Krebs von den Lungen-6, Surfactant protein-D, Lactate dehydrogenase, GAP (gender, age, Physiology) stage, Cardiovascular disease
الوصف: Background Acute exacerbations of idiopathic pulmonary fibrosis are major causes of morbidity and mortality among patients with idiopathic pulmonary fibrosis. However, acute exacerbations remain unpredictable. The aim of this study was to investigate risk factors for acute exacerbations of idiopathic pulmonary fibrosis. Methods We performed a retrospective cohort study of patients with idiopathic pulmonary fibrosis who visited our institutions from January 1999 to September 2014. We investigated risk factors for acute exacerbations in patients with idiopathic pulmonary fibrosis diagnosed retrospectively based on the official 2011 idiopathic pulmonary fibrosis ATS/ERS/JRS/ALAT Update Statement. Results The idiopathic pulmonary fibrosis study cohort included 65 subjects. The median follow-up period was 2.6 years. During follow-up, 24 patients (36.9 %) experienced acute exacerbations. A Kaplan-Meier curve demonstrated that the 1-year, 2-year, and 3-year incidences of acute exacerbation were 9.6, 19.2 and 31.0 %, respectively. Acute exacerbation exerted a significant impact on overall survival among those with the disease. A log-rank test showed that baseline cardiovascular diseases, higher GAP (gender, age, physiology) stage (≥II), higher serum lactate dehydrogenase level (≥180 U/L), higher serum surfactant protein-D level (≥194.7 ng/mL), higher neutrophil (≥1.77 %) and eosinophil (≥3.21 %) percentages in bronchoalveolar lavage fluid samples, and treatment with an immunosuppressive agent after diagnosis were associated with poor acute exacerbation-free probability. In the Cox analysis adjusted for treatment with an immunosuppressive agent, baseline cardiovascular diseases, higher GAP stage (≥II), and higher eosinophil percentage (≥3.21 %) in bronchoalveolar lavage fluid samples were predictors of an acute exacerbation of idiopathic pulmonary fibrosis. Conclusions This study demonstrated that baseline cardiovascular diseases, higher GAP stage (≥II), and higher eosinophil percentage (≥3.21 %) in ...
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8تقرير
مصطلحات موضوعية: Lymphadenopathy, Castleman's disease, Hyaline Vascular, Plasma Cell, CD, HHV-8: Human Hepes Simplex Virus type 8, HIV: Human Immunodeficiency Virus, SECI: South Egypt Cancer Institute, ESR: Erythrocyte Sedimentation Rate, CRP: C-Reactive Protein, DNA: Deoxyribonucleic Acid, CT: Computerized Tomography, LDH: Lactate Dehydrogenase, CD: Castleman's Disease
الوصف: ntroduction: Lymphadenopathy is a common complaint in children. It's defined as abnormality in size or character of lymph node. It may be localized or generalized. It may be part of a group of signs and symptoms or the sole finding and chief complaint. Several reasons can lead to Lymphadenopathy. Castleman's disease is a rare differential diagnosis of generalized or localized Lymphadenopathy. It's a rare lymph proliferative disorder that rarely occurs in children. Case presentation: We reported two cases, the first one was 13 years old female with multicenteric castleman's disease associated with constitutional manifestations and unusual hyaline vascular pathology successfully treated with monthly combination chemotherapy, the second one was 2.5 years old male with unicentric hyaline vascular castleman's disease with unusual young age at presentation successfully treated with surgical excision. Conclusion: Castleman's disease is a rare cause of Lymphadenopathy. Although, it's rare in children, it can occur at any age. Most of cases of multicenteric castleman's disease are plasma cell variant, yet hyaline vascular type may occur. Multicenteric castleman's disease can be successfully treated with monthly combination chemotherapy. Most cases of unicentric castleman's disease are hyaline vascular type. Although, surgical removal of unicentric type is usually sufficient to achieve complete remission, recurrence can still occur.
وصف الملف: application/pdf
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9تقرير
المؤلفون: Wang, Yujiao, Zhang, Yingxin, Jiang, Tianyi, Meng, Jingjing, Sheng, Binbin, Yang, Chunyu, Gao, Chao, Xu, Ping, Ma, Cuiqing
مصطلحات موضوعية: Glycerol, Pseudomonas putida KT2440, Biocatalyst, NAD-independent lactate dehydrogenase, 2-Oxo-carboxylate
الوصف: Background The surplus of glycerol has increased remarkably as a main byproduct during the biofuel’s production. Exploiting an alternative route for glycerol utilization is significantly important for sustainability of biofuels. Results A novel biocatalyst that could be prepared from glycerol for producing 2-oxo-carboxylates was developed. First, Pseudomonas putida KT2440 was reconstructed by deleting lldR to develop a mutant expressing the NAD-independent lactate dehydrogenases (iLDHs) constitutively. Then, the Vitreoscilla hemoglobin (VHb) was heterologously expressed to further improve the biotransformation activity. The reconstructed strain, P. putida KT2440 (Δ lldR )/pBSPPc Gm - vgb , exhibited high activities of iLDHs when cultured with glycerol as the carbon source. This cost-effective biocatalyst could efficiently produce pyruvate and 2-oxobutyrate from dl -lactate and dl -2-hydroxybutyrate with high molar conversion rates of 91.9 and 99.8 %, respectively. Conclusions The process would not only be a promising alternative for the production of 2-oxo-carboxylates, but also be an example for preparation of efficient biocatalysts for the value-added utilization of glycerol.
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10تقرير
المؤلفون: Bosma, Elleke F, van de Weijer, Antonius H P, van der Vlist, Laurens, de Vos, Willem M, van der Oost, John, van Kranenburg, Richard
مصطلحات موضوعية: Bacillus smithii, Thermophile, Lactate dehydrogenase, Sporulation, Pyruvate dehydrogenase, Counter-selection system
الوصف: Background Microbial conversion of biomass to fuels or chemicals is an attractive alternative for fossil-based fuels and chemicals. Thermophilic microorganisms have several operational advantages as a production host over mesophilic organisms, such as low cooling costs, reduced contamination risks and a process temperature matching that of commercial hydrolytic enzymes, enabling simultaneous saccharification and fermentation at higher efficiencies and with less enzymes. However, genetic tools for biotechnologically relevant thermophiles are still in their infancy. In this study we developed a markerless gene deletion method for the thermophile Bacillus smithii and we report the first metabolic engineering of this species as a potential platform organism. Results Clean deletions of the ldhL gene were made in two B. smithii strains (DSM 4216 T and compost isolate ET 138) by homologous recombination. Whereas both wild-type strains produced mainly l -lactate, deletion of the ldhL gene blocked l -lactate production and caused impaired anaerobic growth and acid production. To facilitate the mutagenesis process, we established a counter-selection system for efficient plasmid removal based on lacZ -mediated X-gal toxicity. This counter-selection system was applied to construct a sporulation-deficient B. smithii Δ ldhL Δ sigF mutant strain. Next, we demonstrated that the system can be used repetitively by creating B. smithii triple mutant strain ET 138 Δ ldhL Δ sigF Δ pdhA , from which also the gene encoding the α-subunit of the E1 component of the pyruvate dehydrogenase complex is deleted. This triple mutant strain produced no acetate and is auxotrophic for acetate, indicating that pyruvate dehydrogenase is the major route from pyruvate to acetyl-CoA. Conclusions In this study, we developed a markerless gene deletion method including a counter-selection system for thermophilic B. smithii , constituting the first report of metabolic engineering in this species. The described markerless gene deletion system paves ...
