ABSTRACT
Bacillus subtilis is recognized as a generally-regarded-as-safe strain, and has been widely used in the biosynthesis of high value-added products, including N-acetylneuraminic acid (NeuAc) which is widely used as a nutraceutical and a pharmaceutical intermediate. Biosensors responding to target products are widely used in dynamic regulation and high-throughput screening in metabolic engineering to improve the efficiency of biosynthesis. However, B. subtilis lacks biosensors that can efficiently respond to NeuAc. This study first tested and optimized the transport capacity of NeuAc transporters, and obtained a series of strains with different transport capacities for testing NeuAc-responsive biosensors. Subsequently, the binding site sequence of Bbr_NanR responding to NeuAc was inserted into different sites of the constitutive promoter of B. subtilis, and active hybrid promoters were obtained. Next, by introducing and optimizing the expression of Bbr_NanR in B. subtilis with NeuAc transport capacity, we obtained an NeuAc-responsive biosensor with wide dynamic range and higher activation fold. Among them, P535-N2 can sensitively respond to changes in intracellular NeuAc concentration, with the largest dynamic range (180-20 245) AU/OD. P566-N2 shows a 122-fold of activation, which is 2 times of the reported NeuAc-responsive biosensor in B. subtilis. The NeuAc-responsive biosensor developed in this study can be used to screen enzyme mutants and B. subtilis strains with high NeuAc production efficiency, providing an efficient and sensitive analysis and regulation tool for biosynthesis of NeuAc in B. subtilis.
Subject(s)
N-Acetylneuraminic Acid/metabolism , Bacillus subtilis/metabolism , Promoter Regions, Genetic/genetics , Binding Sites , Biosensing TechniquesABSTRACT
Objective Sialic acid and its derivatives in mammalian cells mainly include N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and deaminoneuraminic acid (KDN), among which Neu5Ac can be catalyzed by cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) for the synthesis of Neu5Gc. In this study, the transcriptional level of CMAH in different tissues of BALB/c mice were determined by relative fluorescence quantitative PCR, to provide a reference for further analysis of Neu5Gc levels in different tissues. Methods The mRNA sequence of CMAH was searched in the NCBI database and specific primers were designed. The mouse β-actin was selected as an internal control, and the transcriptional levels of CMAH in 9 different organ tissues of BALB/c mice were detected by relative fluorescence quantitative PCR using SYBR Green dye. Results Among the 9 mouse organs, the transcriptional level of CMAH in the liver tissue was the highest, which was 2. 46 times higher of that in the spleen, 3. 17 times of the kidney, 5. 14 times of the trachea, 11. 70 times of the lung, 21. 12 times of the myocardium, 31. 37 times of the skeletal muscles, 66. 90 times of the small intestine and 1056. 99 times of the brain tissue, respectively. Conclusions CMAH is transcribed in many organ tissues of mice, and its transcriptional levels vary in a quite wide range.
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Objective To discuss the effect and clinical value of serum sialic acid (SA) and lactadherin(MFG-E8) in the coronary atherosclerotic heart disease(CHD).Methods Selected 295 patients who visited to Wuhan University Renmin's Hospital cardiovascular medicine (from January to June, 2014), and then according to the results of coronary angiography divide the subjects into control group (n =78, the result of coronary angiography completely normal)and coronary heart disease group(n =217, the results of coronary angiography showed that one major coronary stenosis was more than 30％).Detected their serum level of lipid, SA, MFG-E8, hs-CRP index.At last, analyze the differences of SA and MFG-E8 between these groups, and the correlation with the severity of coronary stenosis (symbolize with Gensini score), use the binary logistic regression analysis to research the relationship among the serum level of SA, MFG-E8 and the risk of CHD.Results Compared with the control group, the patients with CHD have a higher level of serum SA and a lower level of MFG-E8 (U =4 402.5, 2 736.0 respectively,P ＜0.001);the AMI group has a higher level of serum SA than the StA and UA groups (U =1 329.0,957.5 respectively, P ＜0.001);the AMI group has a lower level of serum MFG-E8 than the StA groups (U =1517.0, P ＜ 0.001).The spearman correlation analysis showed that the serum SA has a positively correlation with the level of TG, hsCRP and Gensini score, and has a negatively correlated with the level of MFG-E8 (r =0.214, 0.462, 0.378,-0.322 respectively,P ＜0.01);the MFG-E8 has a negatively correlated with the level of TG, hsCRP and Gensini score (r =-0.18,-0.425,-0.564 respectively, P ＜0.01).Binary logistic regression analysis showed that there was a positive correlation between the serum SA and the risk of CHD (OD =4.782, 5.319, 4.179 3.832, 3.648 respectively, P ＜0.001), and the was a negative correlation between the serum MFG-E8 and the risk of CHD (OD =O.118, 0.112, 0.127, 0.135, 0.134 respectively, P ＜ 0.001).Conclusion With the serum SA rised and the MFG-E8 reduced, the risk of coronary heart disease was higher, combined detection these two indexes may be able to diagnose the CHD and assess the degree of stenosis and the risk of CHD.
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Objective To evaluate the performance of serum sialic acid detection kit using enzymic method and investigate the clinical diagnosis value of sialic acid.Methods one hundred and fifty healthy adults were enrolled in this case control study to establish serum SA reference interval.The analytical performance (accuracy,precision,linearity) of serum sialic acid detection kit using enzymic method was assessed.Two hundred and forty patients were classified into different malignant tumor groups according to their pathological types.Serum SA level of each tumor group was compared with that of normal control group.In tumor groups with statistical difference,benign disease groups were further collected.Receiver operating characteristic (ROC) curve and area under curve (AUC) were used to evaluate the diagnostic value of SA compared with other tumor markers.t test,one-way ANOVA,Mann-Whitney U test were used as statistical methods.Results The reference interval of SA was 479 to 715 mg/L.The detection result of 2 level controls was 584 and 1 482 mg/L respectively,which were both within the acceptable limits.The within-lot and between-lot variations of three level samples were both below 5％.There was a good linear correlation (Y =0.995X-0.177,R2 =0.999) between theoretical value and actual detection result in range of 0-1 052 mg/L.The serum level of SA was (757 ± 177),(514 ± 86) and (597 ± 60) mg/L in gastric cancer group,benign disease control group and normal control group respectively,which had statistically significant difference(F =55.2,P ＜ 0.01).The serum level of SA was(659 ± 127) and (545 ± 66) mg/L in colorectal cancer group and benign disease control group respectively,which had statistically significant difference(F =42.8,P ＜ 0.01).The serum level of SA was (738 ± 157) and (672 ± 161) mg/L in colorectal cancer group and benign disease control group respectively,which did not have statistically significant difference(F =26.3,P ＞ 0.05).The AUC of SA was 0.804,0.724,0.755 in gastric cancer group,colorectal cancer group and lung cancer group respectively,which was higher than that of CEA and CA72-4.In gastric cancer group,the sensitivity of SA was higher than that of CEA (59.5％,24.3％).The AUC of SA was 0.791,0.687,0.790 in gastric cancer,colorectal cancer and lung cancer patients with normal CEA serum level respectively.Conclusions Experimental results show that serum sialic acid detection kit using enzymic method has good performance in the precision and linearity.Sialic acid has some value in the diagnosis of gastric cancer and colorectal cancer and could be a good supplement of CEA in screening of cancer.
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Objective To investigate the clinical significance of the serum sialic acid (SA) detection for the diagnosis and therapy monitoring in liver cancer patients.Methods Patients and healthy people of Chinese academy of medical science cancer hospital from January 2011 to October 2012,were enrolled,including 221 liver cancer patients (183 primary hepatic carcinoma patients and 38 metastatic hepatic carcinoma patients),117 benign liver disease patients and 150 healthy people.The concentration of serum SA were tested by ROCHE P800.The intra-assay and inter-assay coefficient of variation (CV) of SA kit were evaluated by use of low and high concentration samples,measured for 5 days and 4 times each day.Receiver operating characteristic (ROC) curve were used to determine the cut-off of SA using data of 183 cases of primary liver cancer and 150 healthy controls.The area under the curve (ROC-AUC) were used to evaluate the diagnostic value of SA.The changes of serum SA level in 103 cases of primary hepatic carcinoma patients were monitored before therapy and at the 1 st day,7 th day,14 th day,1 st month,3rd month,6 th month and 9 th month after treatment.SPSS16.0 was used to analyse the results.Results The intra and inter-day CVs for low level sample were 2.4％ and 3.2％ respectively,and for high level sample were 2.2％ and 3.1％.The cut-off value of the serum SA was 659 mg/L for liver cancer,the sensitivity and specificity was 63.4％ (1 16/183) and 94.7％ (142/150) respectively.The serum SA level of liver cancer group [(726 ± 173) mg/L] was higher than that of liver benign disease patients group [(552 ± 128) mg/ L] and healthy controls group [(599 ± 62) mg/L,U values were 1832.52 and 887.00,P ＜ 0.01].The serum SA level were tracked in 103 cases of primary hepatic carcinoma patients during therapy period.The serum level of SA elevated to [(817 ± 193) mg/L,t =-3.272,P ＜ 0.05] at I st week after treatment and kept at high level until late in 1st month after treatment [(782 ±173) mg/L,t =-2.694,P＜0.05].In the 3rd month,the SA level decreased to that of pretreatment [(662 ± 138) mg/L,t =1.225,P ＞ 0.05].In the 6th months,the SA level declined to [(615 ± 144) mg/L,t =1.999,P ＜0.05],as well as the level of healthy control group.There were 85 cases of hepatic carcinoma patients with decreased SA level compared with that of pretreatment,and the coincidence rate was 82.5％ (85/103),the Kappa value was 0.79.There were 5 cases of patients with hepatic carcinoma relapse after treatment in 9 th months and the SA levels increased significantly to (939 ± 175) mg/L.Conclusion The serum SA has significant values possibly in the diagnosis and therapy monitoring in liver cancer patients.
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Objeetive To evaluate the serum concentration of sialic acid (SA) and High sensitivity C-reactive protein (hs-CRP) in prehypertension patients and possible correlation of the two factors and blood pressure in prehypertension patients.Methods 61 prehypertension patients,70 hypertension patients and 50 healthy controls were included.Lipid profile,hs-CRP,SA,and body mass index (BMI) were estimated in all the groups.Associations between SA and hs-CRP and blood pressure were analyzed by multiple linear regressions.Results The levels of SA and hs-CRP were higher in the prehypertension group than those in the normal group and lower than those in the hypertension group(P ＜ 0.05).There was significant difference in systolic blood pressure (SBP),diastolic pressure (DBP),BMI and HDL among the three groups(P ＜ 0.05).There was significant difference between the prehypertension group and the hypertension group in LDL,TC and FBG.Multiple linear regression showed that FBG,BMI,SA and hs-CRP were correlated with SBP independently while LDL,BMI,SA and hs-CRP were correlated with DBP independently (P ＜ 0.05).Conclusions The findings of the present study suggest that,there is an association between blood pressure and the levels of serum SA and hs-CRP which might be used as screening biomarkers in prehypertension.
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Objective To investigate serum levels of sialic acids(SA)vs carcino-embryonic antigen(CEA)for the diagnosis of digestive tract cancer.Methods Sixty healthy adults,100 patients with malignant digestive tract tumor and those undergoing radical digestive tract tumor surgery were enrolled in this case-control study.Serum SA level was tested.Serum CEA level was measured by electrochemical luminescence.Sensitivity and specificity of SAvs CEA were calculated.Receiver operating characteristic (ROC)curve and area under the curve(ROC-AUC)were used to evaluate the diagnostic value of SA.Results Serum level of SA of patients with digestive malignant tumors was much higher than the normal controls(P ＜0.01),although serum SA of patients undergoing tumor resection showed no difference with the normal controls(P ＞ 0.05).There was significant difference in SA level between the patients who received surgical therapy or not.The sensitivity and specificity of SA in diagnosing digestive tract cancer was 55.00％ and 93.3％,respectively.The false positive rate was 6.7％ in normal subjects.Conclusion Serum SA or CEA testing may be equally useful in the screening,diagnosis,outcome assessment and followup study of digestive tract cancer.
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Theoretical investigations, using semi-empirical potential functions have been carried out to predict the favoured conformations of the terminal dissaccharide fragments of various sialyloligosaccharides. The proposed conformational similarity for these fragments has been correlated to the binding specificity of neuraminidases. These calculations predict that bacterial neuraminidases have a binding site which can accommodate only two sugar residues and virus neuraminidases have a binding site which can accommodate more than two sugar residues.