Noninvasive early diagnosis of colorectal cancer is conducive for patients to reduce the mortality rate and their suffering from diagnosis procedures.One of the most significant methods to proceed noninvasive early diagnosis of colorectal cancer is analysis of stool DNA.Extracting high quality and great quantity of human genomic DNA from stools guarantees diagnosis accuracy.However,the complexity of stool component hinders DNA extraction.Hence,it is crucial to develop highly efficient extraction methods of human genomic DNA from stools for the DNA analysis-based early diagnosis of colorectal cancer.Currently,two kinds of extraction strategies are employed:one is to directly extract total DNA from stools,the other is to enrich exfoliated colonocytes in stools before DNA extraction.This article reviews the advances on these two kinds of extraction techniques and summarizes their applications.

Objective To evaluate the performance of FL-1 macroporous absorption resin for absorption of total flavonoids from Psidium guajava leaf.Methods The concentration of total flavones in Psidium guajava leaf was determined by ultraviolet spectrophotometry and the absorption behavior of FL-1 macroporous adsorption resins to total flavones in Psidium guajava leaf was examined for the adsorption capacity and the volume of solution loaded.Results Optimal absorption of total flavonoids was achieved with the sample:total flavonoids concentration in the solution of 13.22 mg/mL,the ratio of total flavonoids concentration and macroporous adsorption resin was 1∶4,washing with 70% ethanol at the flow velocity of 2 BV/h.Conclusion FL-1 Macroporous absorption resin can be well applicable for enrichment of total flavonoids in Psidium guaijava leaf.

The national essential drug system is designed to solve the problem of covering hospital expenses with medicine revenue, and the problem of difficulty and high expense to see a doctor. This paper investigated whether the national essential drug system worked in three public community hospitals in Beijing. The result showed that the implementation of the program was now well underway, but left some problems. This paper put forward some suggestion for bidding of essential drug, improving the enthusiasm of the medical staff and improving the function of community hospitals.

Early detection and treatment of high-risk adenomas and colorectal cancer (CRC) can reduce mortality of this disease.CRC screening is aimed at minimizing its harm and colonoscopy is presently the gold standard for it.However,colonoscopy needs bowel preparation and is invasive with high risk of intestinal perforation,causing a bad compliance,which is unfavorable to its popularization and application.Recently,non-invasive detection methods for CRC have gone through a rapid development.Tests based on CRC-related biomarkers in fecal and blood samples provide new options for non-invasive CRC screening.However,detection methods for these biomarkers still need further research and improvement because of the complex composition of feces and blood.In the two aspects of fecal tests and blood tests,the progress of recent studies on non-invasive screening methods for CRC was reviewed in this article.

The modern large-scale pyrosequencing technology is a revolution of DNA sequencing.One of the key points in this technology is to get an ATP sulfurylase immobilized on the surface of magnetic beads and with a high activity.Biotinylated ATP sulfurylase can be immobilized on magnetic beads coated with streptavidin through the specific conjunction between biotin and streptavidin, but using chemical modification method to biotinylate ATPS will affect the activity of the enzyme.ATP sulfurylase fused with the carboxyl terminal 87 residues of Escherichia coli biotin carboxyl carrier protein(BCCP87) was expressed in E.coli using fusion expression strategy.Results from Western blot analysis and SDS-PAGE analysis showed that the fusion protein could be biotinylated in vivo, and the molecular mass of the fusion protein was about 64 ku.The biotinylated ATP sulfurylase could be immobilized on the surface of magnetic beads coated with strepavidin, and the immobilized ATPS could be used for quantification of PPi and pyrosequencing.An effective enzyme for the large-scale chip-based pyrosequencing system was supplied.

A method for the real-time polymerase chain reaction ( PCR ) coupled with high specific invader assay to detect single nucleotide polymorphism ( SNP) was established. To reduce the background signal, the amount of flap endonuclease 1 ( FEN1 enzyme ) and wild-type detection probe was optimized. Under the optimum conditions including 0. 05 μmo/L invasive oligonucleotide probe, 0. 125 μmol/L wild-type detection probe, 0. 5 μmol/L mutation detection probe, 0. 25 μmol/L each fluorescence resonance energy transfer (FRET) probe and 1. 5 U FEN1, the background signal of wild-type sample and mutation sample was dramatically decreased and the background interference to the detecting results was thus eliminated. A total of 21 cases of aldehyde dehydrogenase-2*2 ( ALDH2*2 ) , 19 cases of cytochrome p450 2 C19*2 ( CYP2 C19*2 ) and 19 cases of CYP2C19*3 were analyzed with the established method, and the genotypes of ALDH2*2 were 10 cases of GG homozygote, 8 cases of GA heterozygote and 3 cases of AA homozygote; the genotypes of CYP2C19*2 were 9 cases of GG homozygote, 8 cases of GA heterozygote and 2 cases of AA homozygote;and the genotypes of CYP2C19*3 were 18 cases of GG homozygote and 1 case of GA heterozygote. These results were consistent with those by pyrosequencing. The established method was specific, simple, short time-consuming and low cost, and could be used for the detection of SNP genotyping with non-polluting in single closed tube.

Objective The NDRG4 gene methylation in stool is a candidate biomarker for non?invasive diagnosis of colorectal cancer. However, the traditional methods for methylation detection could not be well applied to stool samples due to the low sensitivity and low specificity. The aim of this study was to develop a highly sensitive and specific method for quantifying the methylated NDRG4 gene in stools. Methods Forty one stool samples were collected from 12 colorectal cancer patients, 4 adenoma patients and 25 nor?mal persons. The invasive reaction was combined with real?time PCR and the relative quantification was performed by 2-ΔCT method to develop the highly sensitive and specific methylated DNA detection method, which was used for detecting NDRG4 methylation levels in 41 of stool samples. Results The sensitivity of the method was as low as 10 copies of methylated NDRG4 gene fragments. The specificity was high enough to distinguish 0.01% of methylated fragments from un?methylated fragments and 105 copies of unmethylated NDRG4 fragments gave noamplification signals. The detection results from 41 of stool samples showed that detection rate of the NDRG4 gene in stool from adenoma and colorectal cancer groups had a significant difference comparing to that from the normal group. Conclusion The 2-ΔCT method could accurately quantify the methylation levels of the NDRG4 gene in stool samples, and provide an efficient tool for non?invasive colorectal cancer detection.

Objective To analyze the clinical risk factors of hemorrhagic transformation (HT) of cardiogenic cerebral embolism and the influence of HT on outcome. Methods The clinical data of 115 inpatients were reviewed from May, 2012 to December, 2015. They were di-vided into HT group (n=58) and non-HT group (n=57). The age, anticoagulant therapy, thrombolytic therapy, infarction diameter, diabetes, coronary heart disease, hyperlipidemia, the National Institutes of Health Stroke Scale (NIHSS) score and HAS-BLED score were compared. The risk factors for HT was screened with the multivariate Logistic regression. NIHSS score and Modified Rankin Scale (mRS) score as hos-pitalization, and one month and three months after stroke were compared. Results There were significant difference in NIHSS score (t=-2.991, P=0.003) and HAS-BLED score (t=-2.499, P=0.014), as well as infarction diameter (χ2=8.355, P=0.004) between HT group and non-HT group. NIHSS score (OR=1.127, P=0.027), HAS-BLED score (OR=1.783, P=0.03) and infarction diameter (OR=4.390, P=0.035) were the risk factors for HT. The incidence of HT was less in low-risk group (HAS-BLED score=0-2) than in high-risk group (HAS-BLED score≥3) (χ2=4.643, P=0.031). The NIHSS score as hospitalization, and one month and three months after stroke were all more in HT group than in non-HT group (t>2.387, P<0.05). The mRS score was more in HT group as hospitalization (t=-2.262, P=0.026), but not significant one and three months later (t<1.468, P>0.05). Conclusion HT tends to happen in the patients of cerebral embolism patients after atrial fibril-lation with severe neural function defect, large infarction diameter and high HAS-BLED score. The neural function is poor in those with HT.

Pyruvate phosphate dikinase (PPDK; EC 2.7.9.1) is found in certain microorganisms and plants, and catalyzes the conversion of AMP, PPi and phosphoenolpyruvate (PEP) to ATP, Pi and pyruvate. Using the genomic DNA of Microbispora rosea subsp. aerata as the template, a DNA fragment encoding the gene PPDK was amplified by PCR and inserted into the expression vector pET28a(+), yielding pET28a (+)-PPDK. The E. coli BL21 (DE3) was transformed with the pET28a (+)-PPDK. After inducing with IPTG, the E. coli BL21 (DE3) [pET28a (+)-PPDK] expressed recombinant PPDK fused to an N-terminal sequence of 6-His Tag. The molecular weight of PPDK was estimated to be 101 kD by SDS-PAGE. The PPDK was purified by His * Bind Resin affinity chromatography and ultrafiltration using 10 kD cut-off membrane. The successful application of PPDK in pyrosequencing was also demonstrated.
Actinomyces ; enzymology ; Escherichia coli ; genetics ; metabolism ; Pyruvate, Orthophosphate Dikinase ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis

Seventy-three patients with type 2 diabetes mellitus were divided into atheroselerosis(AS) group and non-AS group according to the intima-media thickness(IMT)of the carotid artery.The plasma visfatin level in AS group was higher than that in non-As group[(44.95±10.14 vs 34.52±9.08)μg/L,P<0.05],and both of them were higher than that of the control [(24.46±7.18)μg/L,both P<0.05 ].The visfatin level Was positively correlated with IMT,waist-to-hip ratio,visceral fat thickness,fasting insulin,and HOMA insulin resistance index.Age,duration of diabetes,HbA_(1C),and visfatin level were the major risk factors for IMT of the carotid artery.