Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.
Bacterial Proteins ; genetics ; Batch Cell Culture Techniques ; Decanoates ; Escherichia coli ; metabolism ; Fermentation ; Glycolipids ; biosynthesis ; Hexosyltransferases ; genetics ; Industrial Microbiology ; methods ; Multigene Family ; Promoter Regions, Genetic ; Pseudomonas aeruginosa ; Rhamnose ; analogs & derivatives ; biosynthesis ; Surface-Active Agents ; metabolism

Objective To investigate the correlation between subclinical hypothyroidism (SCH) and cerebral atherosclerosis burden in patients with large artery atherosclerotic stroke.Methods Consecutive inpatients with acute large artery atherosclerotic stroke were enrolled.SCH was defined as TSH 4.50-10.0 mU/L and serum thyroxine level was normal.Cerebral atherosclerosis burden score was used to evaluate the severity of cerebral atherosclerosis.The total score of 1 or 2 was defined as mild atherosclerosis,and >2 was defined as severe atherosclerosis.Univariate analysis and multivariate logistic regression analysis was used to evaluate the correlation between SCH and severe cerebral atherosclerosis.ResultsA total of 263 patients with large artery atherosclerotic stroke were enrolled,including SCH 62 (23.6%),mild atherosclerosis 119 (45.2%),and severe atherosclerosis 144 (54.8%).The age (63.6±10.9 years vs.60.5±11.4 years;t=2.274,P=0.024),homocysteine (17.10±6.20 μmol/L vs.15.63±5.17 μmol/L;t=2.058,P=0.041),National Institutes of Health Stroke Scale (NIHSS) score (8.0 [5.0-10.0]vs.6.0 [5.0-9.0];Z=2.059,P=0.039),as well as the proportions of patients with hypertension (72.9%vs.58.8%;χ2=5.812,P=0.016),smoking (38.2%vs.26.1%;χ2=4.366,P=0.037),and SCH (30.6%vs.15.1%;χ2=8.610,P=0.003) in the severe cerebral atherosclerosis group were significantly higher than those in the mild cerebral atherosclerosis group.Multivariate logistic regression analysis indicated that SCH was an independent risk factor for severe atherosclerosis in patients with large atherosclerotic stroke (odd ratio 3.345;95%confidence interval 1.692-6.612;P=0.001) after adjusting for age,sex,hypertension,smoking,homocysteine,and NIHSS score.ConclusionSCH is an independent risk factor for severe cerebral atherosclerosis in patients with large atherosclerotic stroke.

OBJECTIVEThis preliminary study aims to investigate the effects of titanium and titanium alloy micro-nano-dimensional topography on the biological behavior of osteoblasts in vitro.

METHODSElectrolytic etching (EE) method was used to produce micro-nano dimensional titanium surfaces. The surfaces were observed to determine their effects on the adhesion, proliferation, cell morphology, and alkaline phosphatase (ALP) activity of osteoblasts.

RESULTSThe surfaces of the titanium and titanium alloy groups exhibited higher adhesion and proliferation of osteoblasts than those of the mechanical group. The titanium surface was covered with a group of cells, a large number of filopodia, and functional particles. The ALP activity of the titanium group was significantly higher than that of the titanium alloy and mechanical groups.

CONCLUSIONEE method in pure titanium and titanium alloy surfaces result in bowl-like nests and nanostructures of different diameters and depths. The diameters of the pure titanium and titanium alloy surfaces range from 30 to 50 μm and 5 to 8 μm, respectively. The former is more conducive to promote the proliferation and differentiation of cells.

Alloys ; Cell Differentiation ; Cell Proliferation ; Dental Etching ; Humans ; Nanostructures ; Osteoblasts ; Prostheses and Implants ; Surface Properties ; Titanium

BACKGROUND:The effects of advanced glycation end products (AGEs) on osteoclast-induced bone resorption is controversial and the underlying mechanisms remain unclear. Most of the studies indicate that AGEs can enhance bone resorption, while some othersshowthe opposite effects.
OBJECTIVE:To investigate the effects of AGEs on osteoclast-induced inorganicmatrixdissolution and organic componentdegradation and the underlying mechanisms.
METHODS:RAW 264.7 cels were induced to generate osteoclasts,and AGEs (50-400 μg/mL) or control-bovine serum albumin (100 μg/mL) was added since the beginning of the induction. The effect of AGEs on bone resorption was evaluated by analyzing the area of resorption pits on the Osteo Assay Surface plates and the expression of cathepsin K. Furthermore, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cels, nuclei per osteoclasts and the expression of integrinανβ3were detected.
RESULTS AND CONCLUSION:The area of resorption pits and expression of cathepsin K in AGEs groups were significantly decreased compared withthecontrol group, and this inhibiting effect became more obvious with the increase of AGEs concentration. TRAP staining also showed that number of TRAP-positivemultinucleated celsand nuclei per osteoclast were significantly reduced in an AGE dose-dependent manner. Quantitative PCR revealed that the expression of integrin ανβ3decreased significantly with the extension of AGEs incubation time. These data indicate that AGEs can exert inhibitory effects on organic and inorganicmatrixdegradation induced by osteoclasts. The underlying mechanism may be involved in the inhibitory effects of AGEs on directed differentiation and cel fusion of osteoclast precursor cels, and migration and adhension of osteoclasts.

Objective This preliminary study aims to investigate the effects of titanium and titanium alloy micro-nanodimensional topography on the biological behavior of osteoblasts in vitro. Methods Electrolytic etching (EE) method was used to produce micro-nano dimensional titanium surfaces. The surfaces were observed to determine their effects on the adhesion, proliferation, cell morphology, and alkaline phosphatase (ALP) activity of osteoblasts. Results The surfaces of the titanium and titanium alloy groups exhibited higher adhesion and proliferation of osteoblasts than those of the mechanical group. The titanium surface was covered with a group of cells, a large number of filopodia, and functional particles. The ALP activity of the titanium group was significantly higher than that of the titanium alloy and mechanical groups. Conclusion EE method in pure titanium and titanium alloy surfaces result in bowl-like nests and nanostructures of different diameters and depths. The diameters of the pure titanium and titanium alloy surfaces range from 30 to 50 μm and 5 to 8 μm, respectively. The former is more conducive to promote the proliferation and differentiation of cells.

Background and objective Radiofrequency ablation (RFA) of lung tumours has recently received much attention for the promising results achieved. Here, to evaluate the value of three-dimensional reconstruction CT in radiofrequen-cy ablation (RFA) of advanced non-small cell lung cancer. Methods Sixty-six cases of advanced non-small ceil lung cancer with 68 lesions (2 patients had 2 lesion treated in one session) were underwent three-dimensional reconstruction CT-giuded percuta-neous RFA therapy. To evaluate short-term therapeutic effect of lung tumors using spiral CT scanning in 1-3 months after RFA to investigate the alterations of minor size and density pre-and post-procedure, and complications, to observe the short-term cura-tive effect. Results Our experiences have shown an initial increase in lesion size at immediate follow-up CT. The density of 64 lesions was lowered (94.1%) and 4 lesion is increased (5.9%) at immediate and one month follow-up CT. SPECT scan findings that 82.4% (56/68) cases of FDG uptake in tumors after RFA with tumor/non-tumor of lower than 2.5 at one month follow-up. The change in treated lesion size over time, radiologically assessed through measurements of the lesions on axial CT scans in the lung window setting no lesions had complete response, 50 lesions (73.5%) had partial response, 2 lesions with stable disease, 6 lesions showed progressive disease at 3 month follow-up CT. SPECT scan findings that 79.4% (54/68) cases of FDG uptake in tumors after RFA with tumor/non-tumor of lower than 2.5 at 3 month follow-up. Conclusion The percutaneons RFA therapy under the guidance of three-dimensionsl reconstruction CT scan is safe and effective, with few complications, and can serve as a new method to the treatment of advanced non-small ceil lung cancers.

BACKGROUND: Lung cancer is the most common malignant tumor in clinic. The prognosis of advanced patients is poor, and the 5-year survival rate is low. Therefore, early diagnosis becomes the key to improve the prognosis of patients. In recent years, with the development of molecular biology technology, aberrant modification of some driver genes, such as methylation, has become an important method for early diagnosis of lung cancer. The purpose of the present work was to quantitatively evaluate the diagnostic value of abnormal hypermethylation in short state homeobox 2 (SHOX2) promoter region in lung cancer by evidence-based medicine. METHODS: We searched MEDLINE, EMBASE, Ovid, Web of Science and CNKI for literatures related to the relationship between SHOX2 gene promoter hypermethylation and lung cancer. The data of SHOX2 promoter hymethylation in the original study were extracted. The diagnostic sensitivity, specificity and area under receiver operating characteristic (ROC) curve of SHOX2 promoter methylation were calculated. RESULTS: Finally, 13 publications were included in this meta-analysis, and due to significant statistical heterogeneity among the studies (P<0.05) the data was pooled by random effect model. The diagnostic sensitivity and specificity of SHOX2 promoter hypermethylation in the diagnosis of lung cancer were 0.75 (95%CI: 0.74-0.77) and 0.89 (95%CI: 0.88-0.91), respectively; The positive likelihood ratio value was 6.75 (4.56-9.99), and the negative predictive value was 0.36 (0.25-0.52); The diagnostic odds ratio was 23.16 (11.34-47.31), and the area under the ROC curve was 0.9. CONCLUSIONS SHOX2 gene promoter hypermethylation is high in serum, broncholavage fluid and pleural effusion of lung cancer patients, which can be used as a biomarker for auxiliary diagnosis of lung cancer.

cis, cis-muconic acid (MA) is an important platform chemical. Now, majority of reported engineered strains are genetically instable, the exogenous genes are expressed under the control of expensive inducer and the components of their fermentation medium are complex, thus large-scale microbial production of MA is limited due to the lack of suitable strains. Hence, it is still necessary to construct novel high-performance strain that is genetically stable, no induction and grows in simple inorganic fermentation medium. In this study, after 3 exogenous genes (aroZ, aroY, catA) for biosynthesis of MA were integrated into previously constructed 3-hydroshikimate producing Escherichia coli WJ060 strain and combinatorially regulated with 3 constitutive promoters with different strengths, 27 engineered strains were constructed. The best engineered strain, E. coli MA30 could produce 1.7 g/L MA in the simple inorganic fermentation medium without induction. To further enhance the production capacity of MA, the mutant library of E. coli MA30 was constructed by genome replication engineering and screened via high-throughput assay. After two-round screening, the new strain, E. coli MA30-G2 with improved production of MA was obtained, and the titer of MA increased more than 8%. Under the condition of 5 L fed-batch fermentation, E. coli MA30-G2 could produce about 11.5 g/L MA. Combinatorial regulation and high-throughput screening provide important reference to microbial production of other bio-based chemicals.
Escherichia coli ; metabolism ; Fermentation ; Industrial Microbiology ; Metabolic Engineering ; Microorganisms, Genetically-Modified ; Promoter Regions, Genetic ; Sorbic Acid ; analogs & derivatives ; metabolism

p-coumaric acid is an important natural phenolic compound with a variety of pharmacological activities, and also a precursor for the biosynthesis of many natural compounds. It is widely used in foods, cosmetics and medicines. Compared with the chemical synthesis and plant extraction, microbial production of p-coumaric acid has many advantages, such as energy saving and emission reduction. However, the yield of p-coumaric acid by microbial synthesis is too low to meet the requirements of large-scale industrial production. Here, to further improve p-coumaric acid production, the directed evolution of tyrosine ammonia lyase (TAL) encoded by Rhodotorula glutinis tal gene was conducted, and a high-throughput screening method was established to screen the mutant library for improve the property of TAL. A mutant with a doubled TAL catalytic activity was screened from about 10,000 colonies of the mutant library. There were three mutational amino acid sites in this TAL, namely S9Y, A11N, and E518A. It was further verified by a single point saturation mutation. When S9 was mutated to Y, I or N, or A11 was mutated to N, T or Y, the catalytic activity of TAL increased by more than 1-fold. Through combinatorial mutation of three types of mutations at the S9 and A11, the TAL catalytic activity of S9Y/A11N or S9N/A11Y mutants were significantly higher than that of other mutants. Then, the plasmid containing S9N/A11Y mutant was transformed into CP032, a tyrosine-producing E. coli strain. The engineered strain produced 394.2 mg/L p-coumaric acid, which is 2.2-fold higher than that of the control strain, via shake flask fermentation at 48 h. This work provides a new insight for the biosynthesis study of p-coumaric acid.
Ammonia-Lyases/genetics* ; Escherichia coli/genetics* ; Propionates ; Rhodotorula ; Tyrosine/genetics*

Lung cancer is the most common malignant tumor and the leading cause of cancer-related death worldwide. Most of the patients have distant metastasis when visiting the doctor, which seriously affects the survival time and quality of life of the patients. With the development of molecular targeted drugs, lung cancer treatment has been transformed from traditional chemotherapy to targeted therapy and precision medicine has been gradually applied in clinical practice, which can make lung cancer patients live longer and have a better quality of life. We present a case of advanced lung cancer patient who presented to Department of Thoracic Surgery of Beijing Haidian Hospital five years ago. We chose the reasonable treatment options though the genetic tests and circulating tumor DNA tests. We summarized the adverse reactions in the whole course of treatment. The comprehensive therapy we utilized, including targeted therapy, chemotherapy, antiangiogenic agents and local radiotherapy, have resulted in our patient with remaining alive. For advanced non-small cell lung cancer with epidermal growth factor receptor (EGFR) mutation positive, individualized treatment was conducted based on precise genotyping and dynamic monitoring, which can not only control the tumor, but also have mild toxic and side effects. The survival time of the patients was prolonged and the quality of life was guaranteed.
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