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BACKGROUND: This study aimed to investigate the expression and clinical value of microRNA miR-486-5p in diagnosing lumbar spinal stenosis (LSS) patients and predicting the clinical outcomes after minimally invasive spinal surgery (MISS) in LSS patients, and the correlation of miR-486-5p with inflammatory responses in LSS patients. METHODS: This study included 52 LSS patients, 46 patients with lumbar intervertebral disk herniation (LDH) and 42 healthy controls. Reverse transcription quantitative PCR was used to detect miR-486-5p expression. The ability of miR-486-5p to discriminate between different groups was evaluated by receiver-operating characteristic analysis. The visual analogue scale (VAS), Oswestry Disability Index (ODI) and Japanese Orthopaedic Association (JOA) scores at 6 months postoperatively were used to reflect the clinical outcomes of LSS patients. Enzyme-linked immunosorbent assay was used to measure the levels of inflammatory factor [interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α)]. The correlation of miR-486-5p with continuous variables in LSS patients was evaluated by the Pearson correlation coefficient. RESULTS: Expression of serum miR-486-5p was upregulated in LSS patients and had high diagnostic value to screen LSS patients. In addition, serum miR-486-5p could predict the 6-month clinical outcomes after MISS therapy in LSS patients. Moreover, serum miR-486-5p was found to be positively correlated with the levels of IL-1ß and TNF-α in patients with LSS. CONCLUSION: miR-486-5p, increased in LSS patients, can function as an indicator to diagnose LSS and a predictive indicator for the clinical outcomes after MISS therapy in LSS patients. In addition, miR-486-5p may regulate LSS progression by modulating inflammatory responses.
Assuntos
Vértebras Lombares , MicroRNAs , Procedimentos Cirúrgicos Minimamente Invasivos , Estenose Espinal , Humanos , Estenose Espinal/cirurgia , Estenose Espinal/genética , Estenose Espinal/sangue , MicroRNAs/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Vértebras Lombares/cirurgia , Idoso , Prognóstico , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Adulto , Interleucina-1beta/sangue , Interleucina-1beta/genéticaRESUMO
The development of temperature-sensitive sensors upgraded by poly(N-isopropylacrylamide) (PNIPAM) represents a significant stride in enhancing performance and tailoring thermoresponsiveness. In this study, an array of temperature-responsive electrochemical sensors modified with different PNIPAM-based copolymer films were fabricated via a "coating and grafting" two-step film-forming technique on screen-printed platinum electrodes (SPPEs). Chemical composition, grafting density, equilibrium swelling, surface wettability, surface morphology, amperometric response, cyclic voltammograms, and other properties were evaluated for the modified SPPEs, successively. The modified SPPEs exhibited significant changes in their properties depending on the preparation concentrations, but all the resulting sensors showed excellent stability and repeatability. The modified sensors demonstrated favorable sensitivity to hydrogen peroxide and L-ascorbic acid. Furthermore, notable temperature-induced variations in electrical signals were observed as the electrodes were subjected to temperature fluctuations above and below the lower critical solution temperature (LCST). The ability to reversibly respond to temperature variations, coupled with the tunability of PNIPAM's thermoresponsive properties, opens up new possibilities for the design of sensors that can adapt to changing environments and optimize their performance accordingly.
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BACKGROUND: 5-hydroxytryptophan (5-HTP), the direct biosynthetic precursor of the neurotransmitter 5-hydroxytryptamine, has been shown to have unique efficacy in the treatment of a variety of disorders, including depression, insomnia, and chronic headaches, and is one of the most commercially valuable amino acid derivatives. However, microbial fermentation for 5-HTP production continues to face many challenges, including low titer/yield and the presence of the intermediate L-tryptophan (L-Trp), owing to the complexity and low activity of heterologous expression in prokaryotes. Therefore, there is a need to construct an efficient microbial cell factory for 5-HTP production. RESULTS: We describe the systematic modular engineering of wild-type Escherichia coli for the efficient fermentation of 5-HTP from glucose. First, a xylose-induced T7 RNA polymerase-PT7 promoter system was constructed to ensure the efficient expression of each key heterologous pathway in E. coli. Next, a new tryptophan hydroxylase mutant was used to construct an efficient tryptophan hydroxylation module, and the cofactor tetrahydrobiopterin synthesis and regeneration pathway was expressed in combination. The L-Trp synthesis module was constructed by modifying the key metabolic nodes of tryptophan biosynthesis, and the heterologous synthesis of 5-HTP was achieved. Finally, the NAD(P)H regeneration module was constructed by the moderate expression of the heterologous GDHesi pathway, which successfully reduced the surplus of the intermediate L-Trp. The final engineered strain HTP11 was able to produce 8.58 g/L 5-HTP in a 5-L bioreactor with a yield of 0.095 g/g glucose and a maximum real-time productivity of 0.48 g/L/h, the highest values reported by microbial fermentation. CONCLUSION: In this study, we demonstrate the successful design of a cell factory for high-level 5-HTP production, combined with simple processes that have potential for use in industrial applications in the future. Thus, this study provides a reference for the production of high-value amino acid derivatives using a systematic modular engineering strategy and a basis for an efficient engineered strain development of 5-HTP high-value derivatives.
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5-Hidroxitriptofano , Engenharia Metabólica , 5-Hidroxitriptofano/genética , 5-Hidroxitriptofano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Glucose/metabolismo , NAD/metabolismo , Neurotransmissores/metabolismo , Serotonina/metabolismo , Triptofano/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Xilose/metabolismoRESUMO
The treatment of diabetes lies in developing novel functional carriers, which are expected to have the unique capability of monitoring blood glucose levels continuously and dispensing insulin correctly and timely. Hence, this study is proposing to create a smart self-regulated insulin delivery system according to changes in glucose concentration. Temperature and glucose dual responsive copolymer microcapsules bearing N-isopropylacrylamide and 3-acrylamidophenylboronic acid as main components were developed by bottom-spray coating technology and template method. The insulinoma ß-TC6 cells were trapped in the copolymer microcapsules by use of temperature sensitivity, and then growth, proliferation, and glucose-responsive insulin secretion of microencapsulated cells were successively monitored. The copolymer microcapsules showed favorable structural stability and good biocompatibility against ß-TC6 cells. Compared with free cells, the biomicrocapsules presented a more effective and safer glucose-dependent insulin release behavior. The bioactivity of secreted and released insulin did not differ between free and encapsulated ß-TC6 cells. The results demonstrated that the copolymer microcapsules had a positive effect on real-time sensing of glucose and precise controlled release of insulin. The intelligent drug delivery system is supposed to mimic insulin secretion in a physiological manner, and further provide new perspectives and technical support for the development of artificial pancreas.
Assuntos
InsulinaRESUMO
Microbial production of l-tryptophan (l-trp) has received considerable attention because of its diverse applications in food additives and pharmaceuticals. Overexpression of rate-limiting enzymes and blockage of competing pathways can effectively promote microbial production of l-trp. However, the biosynthetic process remains suboptimal due to imbalanced flux distribution between central carbon and tryptophan metabolism, presenting a major challenge to further improvement of l-trp yield. In this study, we redistributed central carbon metabolism to improve phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P) pools in an l-trp producing strain of Escherichia coli for efficient l-trp synthesis. To do this, a phosphoketolase from Bifidobacterium adolescentis was introduced to strengthen E4P formation, and the l-trp titer and yield increased to 10.8 g/L and 0.148 g/g glucose, respectively. Next, the phosphotransferase system was substituted with PEP-independent glucose transport, meditated by a glucose facilitator from Zymomonas mobilis and native glucokinase. This modification improved l-trp yield to 0.164 g/g glucose, concomitant with 58% and 40% decreases of acetate and lactate accumulation, respectively. Then, to channel more central carbon flux to the tryptophan biosynthetic pathway, several metabolic engineering strategies were applied to rewire the PEP-pyruvate-oxaloacetate node. Finally, the constructed strain SX11 produced 41.7 g/L l-trp with an overall yield of 0.227 g/g glucose after 40 h fed-batch fermentation in 5-L bioreactor. This is the highest overall yield of l-trp ever reported from a rationally engineered strain. Our results suggest the flux redistribution of central carbon metabolism to maintain sufficient supply of PEP and E4P is a promising strategy for efficient l-trp biosynthesis, and this strategy would likely also increase the production of other aromatic amino acids and derivatives.
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Vias Biossintéticas , Carbono/metabolismo , Escherichia coli , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Triptofano/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Triptofano/genética , Zymomonas/genéticaRESUMO
Various "rich lists" indicate that China now has one of the highest percentages of super rich people worldwide. However, very little is known about their sociopolitical profiles. Based on the annual Hurun China Rich Lists from 2000 to 2018, we created a new dataset of China's super rich that combines information from various sources. Using a multiple correspondent analysis, we reveal that the social spaces of China's super rich are derived from their political, cultural, and social capital. The Chinese super rich are mainly distinguished by their cultural and political capital, while their social capital is not fully independent from political capital. We also identify a division among the super-rich who first appeared on the rich list during different periods. These divisions can inform the understanding of the emergence of the super-rich in an economy that has only recently embraced capitalism and can help predict their evolution in an increasingly glocalized system.
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Capitalismo , Capital Social , China , HumanosRESUMO
L-valine is an essential amino acid and an important amino acid in the food and feed industry. The relatively low titer and low fermentation yield currently limit the large-scale application of L-valine. Here, we constructed a chromosomally engineered Escherichia coli to efficiently produce L-valine. First, the synthetic pathway of L-valine was enhanced by heterologous introduction of a feedback-resistant acetolactate acid synthase from Bacillus subtilis and overexpression of other two enzymes in the L-valine synthetic pathway. For efficient efflux of L-valine, an exporter from Corynebacterium glutamicum was subsequently introduced. Next, the precursor pyruvate pool was increased by knockout of GTP pyrophosphokinase and introduction of a ppGpp 3'-pyrophosphohydrolase mutant to facilitate the glucose uptake process. Finally, in order to improve the redox cofactor balance, acetohydroxy acid isomeroreductase was replaced by a NADH-preferring mutant, and branched-chain amino acid aminotransferase was replaced by leucine dehydrogenase from Bacillus subtilis. Redox cofactor balance enabled the strain to synthesize L-valine under oxygen-limiting condition, significantly increasing the yield in the presence of glucose. Two-stage fed-batch fermentation of the final strain in a 5 L bioreactor produced 84 g/L L-valine with a yield and productivity of 0.41 g/g glucose and 2.33 g/L/h, respectively. To the best of our knowledge, this is the highest L-valine titer and yield ever reported in E. coli. The systems metabolic engineering strategy described here will be useful for future engineering of E. coli strains for the industrial production of L-valine and related products.
Assuntos
Corynebacterium glutamicum , Escherichia coli , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Engenharia Metabólica , Valina/genéticaRESUMO
L-Theanine is a unique non-protein amino acid found in tea plants that has been shown to possess numerous functional properties relevant to food science and human nutrition. L-Theanine has been commercially developed as a valuable additive for use in food and beverages, and its market is expected to expand substantially if the production cost can be lowered. Although the enzymatic approach holds considerable potential for use in L-theanine production, demand exists for developing more tractable methods (than those currently available) that can be implemented under mild conditions and will reduce operational procedures and cost. Here, we sought to engineer fermentative production of L-theanine in Corynebacterium glutamicum, an industrially safe host. For L-theanine synthesis, we used γ-glutamylmethylamide synthetase (GMAS), which catalyzes the ATP-dependent ligation of L-glutamate and ethylamine. First, distinct GMASs were expressed in C. glutamicum wild-type ATCC 13032 strain and GDK-9, an L-glutamate overproducing strain, to produce L-theanine upon ethylamine addition to the hosts. Second, the L-glutamate exporter in host cells was disrupted, which markedly increased the L-theanine titer in GDK-9 cells and almost eliminated the accumulation of L-glutamate in the culture medium. Third, a chromosomally gmasMm-integrated L-alanine producer was constructed and used, attempting to synthesize ethylamine endogenously by expressing plant-derived L-serine/L-alanine decarboxylases; however, these enzymes showed no L-alanine decarboxylase activity under our experimental conditions. The optimal engineered strain that we ultimately created produced ~ 42 g/L L-theanine, with a yield of 19.6%, in a 5-L fermentor. This is the first report of fermentative production of L-theanine achieved using ethylamine supplementation.
Assuntos
Corynebacterium glutamicum/metabolismo , Fermentação , Glutamatos/biossíntese , Engenharia Metabólica/métodos , Trifosfato de Adenosina/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Etilaminas/metabolismo , Ácido Glutâmico/metabolismo , Microbiologia IndustrialRESUMO
A highly sensitive, specific and simple LC-MS/MS method for quantification of capmatinib (INC280) in rat plasma was presented. The LC-MS/MS method was validated in terms of specificity and selectivity, linearity, accuracy and precision, matrix effect, extraction recovery, dilution integrity, carryover and stability as per the US Food and Drug Administration's bioanalytical method validation guideline. The validated assay was applied for quantification of capmatinib from a pharmacokinetic study in rats following oral administration at the doses of 1.0, 3.0 and 9.0 mg/kg. The calibration curve ranges from 1 to 2000 ng/ml with desirable linearity and r2 > 0.99. The intra- and inter-batch accuracies were within 99.24-103.59 and 97.76-102.83% with coefficients of variation 5.08-7.36 and 3.18-4.99%, respectively. No significant interference was observed by endogenous peak at the retention time of capmatinib and IS. The assay was free from any matrix effect and showed precise recovery across the calibration curve range, and samples were stable under all experimental conditions. The validated assay was successfully applied to analyze plasma samples of pharmacokinetic study in rat to determine the concentration of capmatinib. In summary, a novel method for analyzing capmatinib in rat plasma has been successfully validated and is now being utilized for quantification of capmatinib from pre-clinical studies.
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Cromatografia Líquida de Alta Pressão/métodos , Imidazóis/sangue , Espectrometria de Massas em Tandem/métodos , Triazinas/sangue , Administração Oral , Animais , Benzamidas , Imidazóis/administração & dosagem , Imidazóis/farmacocinética , Limite de Detecção , Modelos Lineares , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Triazinas/administração & dosagem , Triazinas/farmacocinéticaRESUMO
Although CRISPR/Cas9-mediated gene editing technology has developed vastly in Escherichia coli, the chromosomal integration of large DNA fragment is still challenging compared with gene deletion and small fragment integration. Moreover, to guarantee sufficient Cas9-induced double-strand breaks, it is usually necessary to design several gRNAs to select the appropriate one. Accordingly, we established a practical daily routine in the laboratory work, involving multiple-step chromosomal integration of the divided segments from a large DNA fragment. First, we introduced and optimized the protospacers from Streptococcus pyogenes in E. coli W3110. Next, the appropriate fragment size for each round of integration was optimized to be within 3-4 kb. Taking advantage of the optimized protospacer/gRNA pairs, a DNA fragment with a total size of 15.4 kb, containing several key genes for uridine biosynthesis, was integrated into W3110 chromosome, which produced 5.6 g/L uridine in shake flask fermentation. Using this strategy, DNA fragments of virtually any length can be integrated into a suitable genomic site, and two gRNAs can be alternatively used, avoiding the tedious construction of gRNA-expressing plasmids. This study thus presents a useful strategy for large DNA fragment integration into the E. coli chromosome, which can be easily adapted for use in other bacteria.
Assuntos
Sistemas CRISPR-Cas , Cromossomos Bacterianos/genética , Fragmentação do DNA , DNA Bacteriano/genética , Escherichia coli/genética , Sequência de Bases , Clonagem Molecular , Deleção de Genes , Edição de Genes , Genes Bacterianos , Plasmídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/genéticaRESUMO
Using a diagonal reference model to analyze data from three waves of Chinese General Social Surveys conducted between 2006 and 2012, we examine how social mobility affects political participation in urban China. We classify political participation into three main categories: voting participation (grassroots elections in the People's Congress and neighborhood committees); voluntary participation (civic activities in social organizations and NGOs); and mixed participation (activities in state corporatism organizations). Our findings demonstrate that there is an asymmetry effect of social mobility in voluntary participation in which the upwardly mobile tend to adapt more to their destinations, while the downwardly mobile tend to adhere more to their origins. But in both voting and mixed participation, political behaviors are more influenced by destination effect. These findings suggest that despite increased civic engagement in NGOs and nonprofit organizations as a mode of voluntary participation, identification with the party apparatus still remains an important factor in explaining the political behaviors of the upwardly mobile in China. When people rise in class status, they tend to be more politically conservative and to build alliances with state agencies by participating in party-sponsored political initiatives.
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Uridine is a kind of pyrimidine nucleoside that has been widely applied in the pharmaceutical industry. Although microbial fermentation is a promising method for industrial production of uridine, an efficient microbial cell factory is still lacking. In this study, we constructed a metabolically engineered Escherichia coli capable of high-yield uridine production. First, we developed a CRISPR/Cas9-mediated chromosomal integration strategy to integrate large DNA into the E. coli chromosome, and a 9.7â¯kb DNA fragment including eight genes in the pyrimidine operon of Bacillus subtilis F126 was integrated into the yghX locus of E. coli W3110. The resultant strain produced 3.3â¯g/L uridine and 4.5â¯g/L uracil in shake flask culture for 32â¯h. Subsequently, five genes involved in uridine catabolism were knocked out, and the uridine titer increased to 7.8â¯g/L. As carbamyl phosphate, aspartate, and 5'-phosphoribosyl pyrophosphate are important precursors for uridine synthesis, we further modified several metabolism-related genes and synergistically improved the supply of these precursors, leading to a 76.9% increase in uridine production. Finally, nupC and nupG encoding nucleoside transport proteins were deleted, and the extracellular uridine accumulation increased to 14.5â¯g/L. After 64â¯h of fed-batch fermentation, the final engineered strain UR6 produced 70.3â¯g/L uridine with a yield and productivity of 0.259â¯g/g glucose and 1.1â¯g/L/h, respectively. To the best of our knowledge, this is the highest uridine titer and productivity ever reported for the fermentative production of uridine.
Assuntos
Escherichia coli , Engenharia Metabólica , Microrganismos Geneticamente Modificados , Uridina/biossíntese , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Loci Gênicos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Óperon , Uridina/genéticaRESUMO
4-Hydroxyisoleucine (4-HIL) exhibits a unique glucose-dependent insulinotropic activity and is a promising candidate for the treatment of diabetes. Direct fermentation of 4-HIL has been recently studied; however, the expected titre and yield were not achieved. In this study, we initially developed a pathway for the synthesis of 4-HIL in an L-isoleucine producer, C. glutamicum YI, but insufficient supply of α-ketoglutarate was a bottleneck for a strong production. Six genes involved in oxaloacetate and α-ketoglutarate branches were overexpressed or deleted, which increased the production of 4-HIL to 5.12â¯g/L but a considerable amount of L-isoleucine still accumulated in the culture. We then dynamically modulated the activity of the α-ketoglutarate dehydrogenase complex (ODHC) by employing L-isoleucine-responsive transcription or attenuation strategies. The best-engineered strain, HIL18, produced 34.21â¯g/L 4-HIL with a negligible accumulation of byproducts, including approximately 0.6â¯g/L L-isoleucine. This study achieved the highest production and yield of 4-HIL, and optimizing the TCA cycle by dynamically modulating the activity of ODHA can be a powerful strategy to balance the carbon flux and achieve efficient production of α-ketoglutarate and derivatives.
Assuntos
Corynebacterium glutamicum , Isoleucina/análogos & derivados , Engenharia Metabólica , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Isoleucina/biossíntese , Isoleucina/genéticaRESUMO
In this study, a uridine and acetoin co-production pathway was designed and engineered in Bacillus subtilis for the first time. A positive correlation between acetoin and uridine production was observed and investigated. By disrupting acetoin reductase/2,3-butanediol dehydrogenasegenebdhA, the acetoin and uridine yield was increased while 2,3-butanediol formation was markedly reduced. Subsequent overexpression of the alsSD operon further improved acetoin yield and abolished acetate formation. After optimization of fermentation medium, key supplementation strategies of yeast extract and soybean meal hydrolysate were identified and applied to improve the co-production of uridine and acetoin. With a consumption of 290.33 g/L glycerol, the recombinant strain can accumulate 40.62 g/L uridine and 60.48 g/L acetoin during 48 h of fed-batch fermentation. The results indicate that simultaneous production of uridine and acetoin is an efficient strategy for balancing the carbon metabolism in engineered Bacillus subtilis. More importantly, co-production of value-added products is a possible way to improve the economics of uridine fermentation.
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Acetoína/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Uridina/biossíntese , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fermentação , Engenharia Metabólica , ÓperonRESUMO
Alpha-ketobutyrate has been widely used in medicine and food additive industry. Because chemical and enzymatic methods are associated with many deficiencies, the recent focus shifted to fermentation for the production of α-ketobutyrate. In this study, a genetically engineered strain THRDΔrhtAΔilvIH/pWSK29-ilvA was constructed, starting from an L-threonine-producing strain, by overexpressing threonine dehydratase (TD), reducing α-ketobutyrate catabolism and L-threonine export. The shake flask cultivation of THRDΔrhtAΔilvIH/pWSK29-ilvA allowed the production of 16.2 g/L α-ketobutyrate. Accumulation of α-ketobutyrate severely inhibited the cell growth. To develop a better TD expression system and avoid the usage of the expensive inducer IPTG, a temperature-induced plasmid pBV220-ilvA was selected to transform the strain THRDΔrhtAΔilvIH for α-ketobutyrate production. The initial temperature was maintained at 35°C to guarantee normal cell growth, and then elevated to 40°C to induce the expression of TD. Under optimized conditions, the α-ketobutyrate titer reached 40.8 g/L after 28 h of fermentation, with a productivity of 1.46 g/L/h and a yield of 0.19 g/g glucose, suggesting large-scale production potential. Biotechnol. Bioeng. 2016;113: 2054-2059. © 2016 Wiley Periodicals, Inc.
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Butiratos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Engenharia Metabólica/métodos , Técnicas de Cultura Celular por Lotes , Butiratos/análise , Escherichia coli/genética , Fermentação , Redes e Vias Metabólicas , Temperatura , Treonina DesidrataseRESUMO
During xylitol fermentation, Candida tropicalis is often inhibited by inhibitors in hemicellulose hydrolysate. The mechanisms involved in the metabolic responses to inhibitor stress and the resistances to inhibitors are still not clear. To understand the inhibition mechanisms and the metabolic responses to inhibitors, a GC/MS-based metabolomics approach was performed on C. tropicalis treated with and without complex inhibitors (CI, including furfural, phenol and acetic acid). Partial least squares discriminant analysis was used to determine the metabolic variability between CI-treated groups and control groups, and 25 metabolites were identified as possible entities responsible for the discrimination caused by inhibitors. We found that xylose uptake rate and xylitol oxidation rate were promoted by CI treatment. Metabolomics analysis showed that the flux from xylulose to pentose phosphate pathway increased, and tricarboxylic acid cycle was disturbed by CI. Moreover, the changes in levels of 1,3-propanediol, trehalose, saturated fatty acids and amino acids showed different mechanisms involved in metabolic responses to inhibitor stress. The increase of 1,3-propanediol was considered to be correlated with regulating redox balance and osmoregulation. The increase of trehalose might play a role in protein stabilization and cellular membranes protection. Saturated fatty acids could cause the decrease of membrane fluidity and make the plasma membrane rigid to maintain the integrity of plasma membrane. The deeper understanding of the inhibition mechanisms and the metabolic responses to inhibitors will provide us with more information on the metabolism regulation during xylitol bioconversion and the construction of industrial strains with inhibitor tolerance for better utilization of bioresource.
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Candida tropicalis/efeitos dos fármacos , Candida tropicalis/metabolismo , Fermentação , Xilitol/metabolismo , Ácido Acético/farmacologia , Candida tropicalis/crescimento & desenvolvimento , Cloretos/farmacologia , Furaldeído/farmacologia , Metaboloma , Metabolômica/métodos , Xilose/metabolismoRESUMO
Steam explosion is the most promising technology to replace conventional acid hydrolysis of lignocellulose for biomass pretreatment. In this paper, a new screw-steam-explosive extruder was designed and explored for xylose production and lignocellulose biorefinery at the pilot scale. We investigated the effect of different chemicals on xylose yield in the screw-steam-explosive extrusion process, and the xylose production process was optimized as followings: After pre-impregnation with sulfuric acid at 80 °C for 3 h, corncob was treated at 1.55 MPa with 9 mg sulfuric acid/g dry corncob (DC) for 5.5 min, followed by countercurrent extraction (3 recycles), decoloration (activated carbon dosage 0.07 g/g sugar, 75 °C for 40 min), and ion exchange (2 batches). Using this process, 3.575 kg of crystal xylose was produced from 22 kg corncob, almost 90 % of hemicellulose was released as monomeric sugar, and only a small amount of by-products was released (formic acid, acetic acid, fural, 5-hydroxymethylfurfural, and phenolic compounds were 0.17, 1.14, 0.53, 0.19, and 1.75 g/100 g DC, respectively). All results indicated that the screw-steam-explosive extrusion provides a more effective way to convert hemicellulose into xylose and could be an alternative method to traditional sulfuric acid hydrolysis process for lignocellulose biorefinery.
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Biomassa , Biotecnologia/métodos , Resíduos Industriais , Xilose/química , Zea mays/química , Carbono/química , Cristalização , Desenho de Equipamento , Concentração de Íons de Hidrogênio , Hidrólise , Íons , Lignina/química , Fenol/química , Polissacarídeos/química , Pressão , Vapor , Ácidos Sulfúricos/química , Temperatura , Fatores de Tempo , Águas ResiduáriasRESUMO
Osteoporotic vertebral compression fracture (OVCF) is a serious complication of osteoporosis, and percutaneous vertebroplasty (PVP) is a major therapeutic method for OVCF. This study aimed to evaluate the clinical efficacy and postoperative complications of robot-assisted targeted PVP for the treatment of OVCF. The data from 202 OVCF patients were analyzed in this study, including 72 cases received traditional PVP (PVP group), 68 cases received robot-assisted PVP (R-PVP group), and 62 cases underwent robot-assisted PVP combined with targeted plugging (R-PVP + TP group). The fluoroscopic exposure conditions, operative duration, lengths of stay, postoperative bone cement leakage, refracture, Visual Analog Scale (VAS) score, and Oswestry Disability Index (ODI) score were obtained and compared between the three groups. The Kaplan-Meier method and logistic regression model were adopted to screen the risk factors related with postoperative refracture. R-PVP and R-PVP + TP group had significantly reduced fluoroscopic frequency and radiation dose, and reduced cement leakage compared with PVP group. R-PVP + TP not only showed more obvious advantages in these aspects, but also had a lower probability of postoperative refracture. In addition, BMD, fracture vertebral distribution, cement leakage, and surgery methods were independent related with refracture. All the results demonstrated robot assistance could improve the application of PVP in the treatment of OVCF, and robot-assisted PVP combined with targeted plugging showed significantly reduced fluoroscopic exposure, bone cement leakage, and rate of postoperative refracture. BMD, fracture vertebral distribution, cement leakage, and operation methods were identified as four risk factors for the onset of refracture after PVP.
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Fraturas por Compressão , Cifoplastia , Fraturas por Osteoporose , Procedimentos Cirúrgicos Robóticos , Robótica , Fraturas da Coluna Vertebral , Vertebroplastia , Humanos , Fraturas por Compressão/cirurgia , Fraturas por Compressão/complicações , Fraturas por Compressão/tratamento farmacológico , Cimentos Ósseos/uso terapêutico , Vertebroplastia/efeitos adversos , Vertebroplastia/métodos , Fraturas da Coluna Vertebral/diagnóstico por imagem , Fraturas da Coluna Vertebral/cirurgia , Fraturas da Coluna Vertebral/tratamento farmacológico , Cifoplastia/efeitos adversos , Cifoplastia/métodos , Estudos Retrospectivos , Procedimentos Cirúrgicos Robóticos/métodos , Fraturas por Osteoporose/cirurgia , Fraturas por Osteoporose/complicações , Fraturas por Osteoporose/tratamento farmacológico , Resultado do Tratamento , Fatores de RiscoRESUMO
Pseudouridine is the most abundant modified nucleoside found in non-coding RNA and is widely used in biological and pharmaceutical fields. However, current methods for pseudouridine production suffer from drawbacks such as complex procedures, low efficiency and high costs. This study presents a novel enzymatic cascade reaction route in Escherichia coli, enabling the whole-cell catalytic synthesis of pseudouridine from uridine. Initially, a metabolic pathway was established through plasmid-mediated overexpression of endogenous pseudouridine-5-phosphase glycosidase, ribokinase, and ribonucleoside hydrolase, resulting in the accumulation of pseudouridine. Subsequently, highly active endogenous ribonucleoside hydrolase was screened to enhance uridine hydrolysis and provide more precursors for pseudouridine synthesis. Furthermore, modifications were made to the substrates and products transport pathways to increase the pseudouridine yield while avoiding the accumulation of by-product uridine. The resulting recombinant strain Ψ-7 catalyzed the conversion of 30 g/L uridine into 27.24 g/L pseudouridine in 24 h, achieving a conversion rate of 90.8% and a production efficiency of 1.135 g/(L·h). These values represent the highest reported yield and production efficiency achieved by enzymatic catalysis methods to date.
Assuntos
Escherichia coli , Pseudouridina , Pseudouridina/genética , Pseudouridina/química , Pseudouridina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Uridina/genética , Uridina/química , Uridina/metabolismo , Catálise , Hidrolases/metabolismoRESUMO
In order to study the multi-mode damage and fracture mechanisms of thin-walled tubular parts with cross inner ribs (longitudinal and transverse inner ribs, LTIRs), the Gurson-Tvergaard-Needleman (GTN) model was modified with a newly proposed stress state function. Thus, tension damage and shear damage were unified by the new stress state function, which was asymmetric with respect to stress triaxiality. Tension damage dominated the modification, which coupled with the shear damage variable, ensured the optimal prediction of fractures of thin-walled tubular parts with LTIRs by the modified GTN model. This included fractures occurring at the non-rib zone (NRZ), the longitudinal rib (LIR) and the interface between the transverse rib (TIR) and the NRZ. Among them, the stripping of material from the outer surface of the tubular part was mainly caused by the shearing of built-up material in front of the rollers under a large wall thickness reduction (ΔT). Shear and tension deformation were the causes of fractures occurring at the NRZ, while axial tension under a large TIR interval (l) mainly resulted in fractures on LIRs. Fractures at the interface between the TIR and NRZ were due to the shearing applied by rib grooves and radial tension during the formation of ribs. This study can provide guidance for the manufacturing of high-performance aluminum alloy thin-walled tubular components with complex inner ribs.