Cloning and prokaryotic expression analysis of AlCMK from Atractylodes lancea / 药学学报

4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK) was one of the key enzymes in the methylerythritol-4-phosphate (MEP) pathway to generate terpenoids. In this study, based on the transcriptome data of Atractylodes lancea, the sequence of the CMK gene was cloned, named AlCMK (GenBank accession number OM283293). The results showed that AlCMK contains a 1 230 bp open reading frame (ORF) encoding 409 amino acids. The deduced protein had a theoretical molecular weight of 44 752.53 and an isoelectric point of 6.67. Transmembrane structure analysis showed that there was no transmembrane structure, and the secondary structure of AlCMK was predicted to be mainly composed of random coil. Homologous alignment revealed that AlCMK shared high sequence identity with the CMK proteins of Tanacetum cinerariifolium, Osmanthus fragrans, Eucommia ulmoides, Lonicera japonica and Salvia miltiorrhiza. Phylogenetic analysis indicated that AlCMK protein had the higher homology with CMK protein of Compositae. The pET-32a-AlCMK prokaryotic expression vector was constructed and a fusion protein with molecular mass of about 65 kDa was expressed in the E. coli BL21 (DE3). The qRT-PCR was used to analyze the expression pattern of AlCMK gene in different tissues and after MeJA treatment. Meanwhile, the enzyme activity was determined by ELISA kit. The results showed that AlCMK gene was tissue-expressed in different origins and its expression was induced by MeJA, and the results of the enzyme activity assay showed that the AlCMK enzyme activity in different regions was higher in the leaves. The subcellular localization showed that AlCMK was located in the chloroplast. This study provides a reference for further elucidating the biological function of AlCMK gene in terpenoid synthesis pathway in Atractylodes lancea.

CRISPR/Cas-mediated DNA base editing technology and its application in biomedicine and agriculture / 生物工程学报

In recent years, the genome editing technologies based on the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) have developed rapidly. The system can use homologous directed recombination (HDR) to achieve precise editing that it medicated, but the efficiency is extremely low, which limits its application in agriculture and biomedical fields. As an emerging genome editing technology, the CRISPR/Cas-mediated DNA base editing technologies can achieve targeted mutations of bases without generating double-strand breaks, and has higher editing efficiency and specificity compared with CRISPR/Cas-mediated HDR editing. At present, cytidine base editors (CBEs) that can mutate C to T, adenine base editors (ABEs) that can mutate A to G, and prime editors (PEs) that enable arbitrary base conversion and precise insertion and deletion of small fragments, have been developed. In addition, glycosylase base editors (GBEs) capable of transitioning from C to G and double base editors capable of editing both A and C simultaneously, have been developed. This review summarizes the development, advances, advantages and limitations of several DNA base editors. The successful applications of DNA base editing technology in biomedicine and agriculture, together with the prospects for further optimization and selection of DNA base editors, are discussed.

Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5'-cytidine monophosphate production / 生物工程学报

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.

Effect of p53 mutation mediated by APOBEC3B on NSCLC metastasis / 中国医师杂志

Objective APOBEC3B (A3B) is an important member of the apolipoprotein B mRNA editing enzyme catalytic polypeptide (APOBEC) family.This study aimed to investigate its important role in the metastasis of small cell lung cancer (NSCLC).Methods The statistical relationship between A3 B and clinicopathological data was analyzed in 249 cases of NSCLC.Sanger sequencing was used to detect mutations in exon 5,6,7 and 8 of P53 in 74 cases of lung cancer.A3B overexpression cell line was constructed in human lung adenocarcinoma cells HCC827 to observe the change of cell migration and metastasis capacity.Results A3B was highly expressed in NSCLC tissues compared with normal lung tissues.The expression of A3B was closely related to the lymph node metastasis of NSCLC and the mutation rate of p53 was positively correlated with the expression of A3B.In vitro experiment,it showed enhanced migration and increased metastatic potential in cells after overexpression of A3B.Conclusions A3B-mediated mutations in P53 may play a key role in the metastasis of NSCLC.

The clinical effect of citicoline sodium combined with Shenxiong glucose in the treatment of senile hypertensive cerebral infarction in the elders / 中国医师杂志

Objective To investigate the clinical therapeutic effect of citicoline sodium combined with Shenxiong glucose in the treatment of senile hypertensive cerebral infarction in the elders.Methods 80 elderly patients with senile hypertensive cerebral infarctiontreated in our hospital were selected and randomly divided into the single treatment group and the combination treatment group,40 cases in each group.Both groups received the routine treatment.The single treatment group additionally received Shenxiong glucose injection (100 ml/d),while patients in the combination treatment group additionally received Shenqiong glucose injection combined with citicoline sodium intravenous infusion therapy (0.5 g/d),both groups were treated for 2 weeks.The levels of serum inflammatory factors,the neurological deficit score,the cognitive function score were compared and analyzed before and after treatment between two groups.Results After systemic treatment,the blood pressure and blood lipid levels of two groups were significantly improved,but there was no significant difference between the two groups (P ＞ 0.05);The serum levels of interleukin (IL)-6,IL-8 and tumor necrosis factor-α (TNF-α) of the combination treatment group improved more significantly (P ≤0.05).After treatment,the oxidative stress indexes were significantly improved in the two groups (P ≤ 0.05).The content of malondialdehyde (MDA) was decreased,while the superoxide dismutase (SOD) activity and nitric oxide (NO) content were increased significantly (P ≤ 0.05);and the improvement degree in the combination treatment group was better than in the single treatment group (P ≤0.05).The degree of improvement in the Modified Edinburgh-Scandinavia Stroke Scale (MESSS) and Hasegawa Dementia Scale (HDS) scores of the combination treatment group was more significant than those in the single treatment group (P ≤ 0.05).The total effective rate of the combination treatment group was 92.5％,which was significantly higher than that of the single group (75.0％),with statistically significant difference (P ≤ 0.05).No obvious adverse reactions happened in two groups during treatment.Conclusions Combination of citicoline sodium and shenxiang glucose on the basis of routine treatment can significantly reduce oxidative stress and inflammation levels,promote the recovery of neurological and cognitive functions,and improve the clinical efficacy and safety.It is worth popularizing and applying in the clinical treatment of senile hypertensive cerebral infarction.

Novel fluoronucleoside analog NCC inhibits lamivudine-resistant hepatitis B virus in a hepatocyte model

ABSTRACT Antiviral drug resistance is the most important factor contributing to treatment failure using nucleos(t)ide analogs such as lamivudine for chronic infection with hepatitis B virus (HBV). Development of a system supporting efficient replication of clinically resistant HBV strains is imperative, and new antiviral drugs are needed urgently to prevent selection of drug-resistant HBV mutants. A novel fluorinated cytidine analog, NCC (N-cyclopropyl-4′-azido-2′-deoxy-2′-fluoro-β-d-cytidine), was recently shown to strongly inhibit human HBV in vitro and in vivo. This study was designed to evaluate the antiviral activity of NCC against lamivudine-resistant HBV. We generated a stable cell line encoding the major pattern of lamivudine-resistant mutations rtL180M/M204V and designated it "HepG2.RL1". Immuno-transmission electron microscopic examination and enzyme-linked immunosorbent assay were used to detect secretion of HBV-specific particles and antigens. Quantification of extracellular DNA and intracellular DNA of HepG2.RL1 cells by quantitative real-time polymerase chain reaction revealed >625-fold and >5556-fold increases in the 50% inhibitory concentration of lamivudine, respectively, compared with that for the wild-type virus. The results showed that NCC inhibited DNA replication and HBeAg production in wild-type or lamivudine-resistant HBV in a dose-dependent manner. In conclusion, screening for antiviral compounds active against lamivudine-resistant HBV can be carried out with relative ease using hepG2.RL1 cells. NCC is a potential antiviral agent against wild-type HBV and clinical lamivudine-resistant HBV and deserves evaluation for the treatment of HBV infection.

HPLC Method for Determination of Seven Components in Holotrichia diomphalia Larvae / 中国药学杂志

OBJECTIVE: To establish a quantitative method for determining the contents of uracil, cytidine, hypoxanthine, xanthine, uridine, inosine, and guanosine in Holotrichia diomphalia Larvae by HPLC. METHODS: The HPLC analysis was performed on Waters HSS T3 column(4.6 mm×250 mm, 5 μm). The mobile phase was composed of acetonitrile(A) and water(B), and gradient elution was carried out. The flow rate was 1.0 mL•min-1. The column temperature was maintained at 30 ℃. The detection wavelength was 260 nm. RESULTS: The correlation coefficients of the seven components ranged from 0.999 1 to 1.000 0. The average recovery rates(n=6) were between 91.2% and 97.4%, and the RSDs were between 1.5% and 2.3%. CONCLUSION: The proposed method is simple, accurate and reliable, thus providing basis for comprehensive quality control of Holotrichia diomphalia Larvae.

Expression of activation-induced cytidine deaminase in renal tissues of patients with systemic lupus erythematosus and its clinical significance / 第二军医大学学报

Objective To explore the expression of activation-induced cytidine deaminase (AICDA) in renal tissues of patients with systemic lupus erythematosus (SLE) and its clinical significance. Methods The renal biospy tissues from 73 patients with SLE and 36 patients with primary membranous nephropathy, who underwent biopsy between Mar. 2013 to Jun. 2017 in Central South University Xiangya School of Medicine Affiliated Haikou Hospital, were collected. Immunohistochemical method was used to detect the expression of AICDA in renal tissues. The correlations between the expression level of AICDA and the clinicopathological parameters, including pathological classification, system damage, SLE disease activity index (DAI) score and treatment outcome of SLE patients were analyzed. Results The expression level of AICDA in renal tissues of SLE patients was significantly higher than that of primary membrane nephropathy patients (6.12±2.47 vs 3.33±1.91, P0.05). The expression levels of AICDA were significantly higher in renal tissues of SLE patients with oral ulcer, interstitial pneumonia, nervous system damage, arthritis, blood system damage or serositis than those in the patients without above symptoms (7.02±2.14 vs 4.17±1.97, 7.86±2.39 vs 4.98±1.76, 9.83±1.34 vs 5.39±1.92, 6.88±2.04 vs 2.93±1.21, 7.51±1.81 vs 3.70±1.23, and 7.29±2.33 vs 5.34±2.29; all P0.05). Conclusion AICDA is related to the occurrence and development of SLE, and it is expected to bring new targets for the treatment of SLE.

The influence of genetic variation of cytidine deaminase on hand-foot syndrome among colorectal cancer patients treated with capecitabine-based adjuvant chemother-apy regimens / 中国肿瘤临床

Objective:To investigate the association between grade 3 hand-foot syndrome(HFS)in colorectal cancer(CRC)patients treated with capecitabine and variation of cytidine deaminase(CDA)genes.Methods:The polymorphisms of the key gene CDA in-volved in capecitabine metabolism were genotyped and 149 CRC patients were included in this study.The association between these polymorphisms and susceptibility to HFS were analyzed.Additionally,peripheral blood mononuclear cells(PBMCs)of 91 CRC patients were collected for mRNA expression analysis, and the levels of mRNA expression according to different CDA genotypes were com-pared.Results:The prevalence of the polymorphism-451G>A,which is located in the promoter region of CDA,were correlated with HFS. The results were as follows: GG genotype, 109 cases (73.15%); GA genotype, 38 cases (25.50%); and AA genotype, 2 cases (1.36%).The minor allele frequency of-451G>A was 0.14.The distribution of the three genotypes were in accordance with Hardy-Weinberg Equilibrium(P=0.516).Logistic analysis indicated that GA/AA genotypes were associated with grade 3 HFS(odds ratio=2.53, P=0.011).Additionally,another insert polymorphism-33delC located in the promoter region of CDA was in linkage disequilibrium with-451G>A (D'=0.92). Of the 91 PBMC mRNA expression analyses, the GA/AA genotype of-451G>A was associated with higher CDA mRNA expression compared with GG genotypes(4.01±0.53 vs.3.13±0.61,P<0.001).Conclusions:The polymorphism-451G>A of CDA may influence occurance of grade 3 HFS induced by capecitabine by influencing CDA mRNA expression.

Effect of Recombinant Human Growth Hormone Add on Therapy on Acute Stroke Outcome

Recombinant human growth hormone (rhGH) administration stimulate the secretion of the brain insulin-like growth factor-1 (IGF-1) concentration and IGF-1 is a pleiotropic neurotropic peptide to exert beneficial effect for the injured brain tissues. Citicoline (cytidine-59-diphosphocholine; CDP-choline) is well known to improve neurological outcome in acute stroke. This study aimed to evaluate whether rhGH can potentiate citicoline effect on functional recovery in acute stroke patient. Thirty patients were enrolled. Ten patients were treated with rhGH subcutaneous injection for 6 months on top of citicoline for 6 weeks (GH6 group), and 10 patients for 3 months (GH3 group) with 6 weeks of citicoline treatment as well, and final 10 patients only with citicoline (control group). Functional outcome was determined by Korean modified Barthel Index (K-MBI) and modified Rankin Scale (mRS) at baseline and 6 months after treatment. Seven and 4 patients withdrew from GH6 and GH3 group, respectively. Final 3 patients in GH6 group, 6 patients in GH3 group and 10 patients in control group were analyzed. The K-MBI, and mRS scores from all 3 groups increased in 6 months compared to baseline in intra-group comparison. In inter-group comparison, however, GH6 but not GH3 showed statistically significant improvement compared to control. Administration of rhGH for 6 months on top of 6-week citicoline treatment resulted in further improvement in K-MBI and mRS in acute stroke patients. Further studies in increasing injection dose or injection period is needed.

Expression and potential clinical significance of cytidine triphosphate: phosphocholine cytidylyltransferase-α in oral squamous cell carcinoma / 中华口腔医学杂志

Objective@#To examine the expression and potential clinical significance of CCT (cytidine triphosphate: phosphocholine cytidylyltransferase)-α in oral squamous cell carcinoma (OSCC).@*Methods@#Fifty-eight OSCC and paired adjacent non-malignant epithelia samples (between May 2016 and July 2016) were obtained from dental center, Second Xiangya Hospital, Central South University. CCT-α expression was examined by immunohistochemistry. The relationship between CCT-α and clinicopathological features of OSCC patients was analyzed. Quantitative real-time PCR and Western blot were performed to measure the expression of CCT-α mRNA and protein level in several OSCC cell line and two normal oral epithelial cell line.@*Results@#Immunohistochemistry showed that CCT-α positive staining was found in cell nuclear of OSCC cells and adjacent epithelial cells. CCT-α was positively expressed in OSCC, which was significantly higher than that adjacent to carcinoma tissues (P=0.000). The expression of CCT-α in oral squamous cell carcinoma was correlated with smoking, alcohol consumption, tumor size, differentiation degree and lymph node metastasis. The expression level of CCT-α protein was significantly increased in patients with a history of smoking and alcohol consumption (P=0.001, P=0.004). With the increase of tumor diameter, the expression of CCT-α protein was significantly increased (P=0.005). According to histopathological grade, the lower the degree of tumor differentiation, the higher the expression level of CCT-α protein (P=0.000). The expression of CCT-α protein was significantly higher in patients with lymph node metastasis compared with no lymph node metastasis (P=0.000). Quantitative real-time PCR results showed the CCT-α mRNA expression level was significantly higher in OSCC cells than that in normal oral epithelial cells (P=0.016). The protein expression level of CCT-α was significantly higher in OSCC cells than that in normal oral epithelial cells.@*Conclusions@#CCT-α may play a critical role in the carcinogenesis and development of OSCC.

Analysis of transcriptional levels of CMAH in different organ tissues of mice / 中国比较医学杂志

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.

Epigenetic modification by 5-Aza/TSA treatment induces loss of B-cell phenotype in B-cell derived non-Hodgkin lymphoma / 中国病理生理杂志

AIM:To investigate influence of demethylation/acetylation by 5-Aza-2'-deoxycytidine/trichostatin A(5-Aza/TSA)treatment on B-cell specific phenotype of non-Hodgkin lymphoma cells.METHODS:CD19 promoter-driven reporter with NEO cassette was constructed to realize transfection and stable selection of Hodgkin and non -Hodgkin lymphoma cells.The exogenous CD19 promoter activity in both cell line clusters with and without 5-Aza/TSA treatment was detected and compared.The B-cell specific expression profiling in Eμ-myc transgenic mouse model developed lymphoma was isolated and identified.The effects of 5-Aza/TSA treatment on B-cell specific phenotype were analyzed.RESULTS:Epigenetic modification via 5-Aza/TSA repressed B-cell specific phenotype in B-cell-derived non-Hodgkin lymphoma cells. CONCLUSION:Epigenetic modification of pivotal master repressor genes plays an essential role in B -cell phenotype of both human and murine developed B-cell non-Hodgkin lymphoma cells.

Effect of citicoline on mild cognitive impairment in patients with middle cerebral artery stenosis / 中华老年心脑血管病杂志

Objective To study the effect of citicoline on mild cognitive impairment MCI) in pa tients with middle cerebral artery stenosis (MCAS).Methods Eighty-six MCAS patients with MCI were divided into citicoline group (n=44) and control group (n=42).The patients in citicoline group were treated with citicoline (0.2 g,3 times a day) for 6 months on the basis of conventional treatment.Cerebrovascular reserve,PI,BHI and Vm between the two groups were compared by transcranial Doppler ultrasonography after treatment.MCI between the two groups was assessed according to the MoCA after treatment.Results The rate of cerebrovascular reserve,PI,BHI and Vm were significantly higher in citicoline group than in control group (13.59％± 1.16％ vs 7.61％±1.12％,P＜0.01;0.51±0.16 vs 0.58±0.12,P＜0.05;1.36±0.08 vs 0.74±0.11,P＜ 0.01;32.63％ ±2.32％ vs 16.92％ ± 1.68％,P＜0.05).The total MoCA score,attention,language,visuospatial and executive function,abstract,naming,orientation and memory were significantly higher in citicoline group than in control group (P＜0.01).Conclusion Early citicoline treatment can improve cerebrovascular reserve and alleviate MCI in MCAS patients.

IFNα induces cytidine/uridine monophosphate kinase 2 expression in hepatocarcinoma cell line to activate macrophages of mice / 基础医学与临床

Objective To explore the role of cytidine/uridine monophosphate kinase 2( CMPK2) in the immune-mediated antitumor effect of IFNα in hepatocellular carcinoma. Methods RT-qPCR and Western blot were used to analyze the expression of CMPK2 in Huh7 after the treatment of IFNα. The CMPK2 overexpressing Huh7 cells were generated by stably infecting with lentivirus. The ATP level in the cells and the supernatant of CMPK2 overexpress-ing Huh7 cells were measured by CellTiter-Glo ATP fluorescence assay. RT-qPCR was applied to test the expression of inflammatory cytokines in macrophages under the treatment of the supernatant of CMPK2 overexpress-ing Huh7 cells. Results The transcription and protein level of CMPK2 were significantly enhanced after the treat-ment of IFNα for 6 hours ( P＜0.01) . CMPK2 increased the ATP level in the cells and supernatant of Huh7 cells ( P＜0.01) . The supernatant of CMPK2 overexpressing Huh7 cells activated the expression of IL1β, IL6 and CCL5 in macrophages( P＜0.01) . Conclusions IFNα increases the expression of CMPK2 in Huh7 cells to activate the expression of inflammatory cytokines in macrophages.

Construction of Mycobacterium tuberculosis cdd knockout and evaluation of invasion and growth in macrophages

Cytidine deaminase (MtCDA), encoded by cdd gene (Rv3315c), is the only enzyme identified in nucleotide biosynthesis pathway of Mycobacterium tuberculosis that is able to recycle cytidine and deoxycytidine. An M. tuberculosis knockout strain for cdd gene was obtained by allelic replacement. Evaluation of mRNA expression validated cdd deletion and showed the absence of polar effect. MudPIT LC-MS/MS data indicated thymidine phosphorylase expression was decreased in knockout and complemented strains. The cdd disruption does not affect M. tuberculosis growth both in Mid- dlebrook 7H9 and in RAW 264.7 cells, which indicates that cdd is not important for macrophage invasion and virulence.

Overexpression of Uridine-Cytidine Kinase 2 Correlates with Breast Cancer Progression and Poor Prognosis / 한국유방암학회지

PURPOSE: Uridine-cytidine kinase (UCK) 2 is a rate-limiting enzyme involved in the salvage pathway of pyrimidine-nucleotide biosynthesis. Recent studies have shown that UCK2 is overexpressed in many types of cancer and may play a crucial role in activating antitumor prodrugs in human cancer cells. In the current study, we evaluated the potential prognostic value of UCK2 in breast cancer. METHODS: We searched public databases to explore associations between UCK2 gene expression and clinical parameters in patients with breast cancer. Gene set enrichment analysis (GSEA) was performed to identify biological pathways associated with UCK2 gene expression levels. Survival analyses were performed using 10 independent large-scale breast cancer microarray datasets. RESULTS: We found that UCK2 mRNA expression was elevated in breast cancer tissue compared with adjacent nontumorous tissue or breast tissue from healthy controls. High UCK2 levels were correlated with estrogen receptor negativity (p<0.001), advanced tumor grade (p<0.001), and poor tumor differentiation (p<0.001). GSEA revealed that UCK2-high breast cancers were enriched for gene sets associated with metastasis, progenitor-like phenotypes, and poor prognosis. Multivariable Cox proportional hazards regression analyses of microarray datasets verified that high UCK2 gene expression was associated with poor overall survival in a dose-response manner. The prognostic power of UCK2 was superior to that of TNM staging and comparable to that of multiple gene signatures. CONCLUSION: These findings suggest that UCK2 may be a promising prognostic biomarker for patients with breast cancer.

Simultaneous determination of five nucleosides in Houtoujun Tablets by RP-HPLC / 中成药

AIM To establish an RP-HPLC method for the simultaneous content determination of five nucleosides in Houtoujun Tablets (mycelia of Hericium erinaceum).METHODS The analysis of aqueous extract of this drug was performed on a 26 ℃ thermostatic Zorbax SB-Aq C18 column (250 mm ×4.6 mm,5 μm),with the mobile phase comprising of methanol-0.1％ phosphoric acid flowing at 0.5 mL/min in a gradient elution manner,and the detection wavelength was set at 260 nm.RESULTS Cytidine,adenine,uridine,adenosine and guanosine showed good linear relationships within the ranges of 0.007-0.169,0.005-0.115,0.032-0.797,0.032-0.792 and 0.004-0.103 μg,whose average recoveries (n =6) were 99.4％ (RSD =2.0％),98.3％ (RSD =1.8％),98.0％ (RSD =2.8％),101.4％ (RSD =1.9％) and 101.7％ (RSD =1.2％),respectively.CONCLUSION The contents of five nucleosides in Houtoujun Tablets from different manufacturers exhibit obvious differences,to which we should pay attention.

Application of Sanger sequencing technology in detection of gemcitabine resistance sensitive gene CDA / 国际检验医学杂志

Objective To analyze the polymorphic site mutation of gemcitabine resistant sensitive gene cytidine deaminase (CDA),and to investigate the applicability of the Sanger detection technology in clinical sample detection with the real-time fluores-cence quantitative PCR as the reference.Methods The peripheral blood samples from 255 cases of stage ⅢB to Ⅳ non small-cell lung cancer(NSCLC)were randomly selected for extracting DNA.The G208A and A79C mutation situation of CDA gene was re-spectively detected by qPCR and Sanger sequencing technology.Results The success rate for detecting the CDA gene mutation in peripheral blood sample in 255 cases of stage ⅢB and Ⅳ NSCLC were 98.04%(250/255)by the Sanger sequencing method and 97.25%(248/255)by qPCR.With the qPCR as the reference,the sensitivity and specificity of Sanger sequencing method was 92.50% and 99.52% respectively.All mutation types detected by two methods were fully consistent.Conclusion Adopting the Sanger sequencing method can effectively detect drug resistance gene CDA mutation,realizes the mutation typing,can accurately, high throughput and efficiently detect CDA mutation site,which provides an effective detection method for gemcitabine sensitive medication of NSCLC.

Epstein-Barr virus infection of infants: implications of early age of infection on viral control and risk for Burkitt lymphoma / Infección en lactantes por virus de Epstein-Barr: implicaciones de la infección a temprana edad sobre el control viral y el riesgo de linfoma de Burkitt

Since its first description by Denis Burkitt, endemic Burkitt's lymphoma (BL), the most common childhood cancer in sub-Saharan Africa, has led scientists to search for clues to the origins of this malignancy. The discovery of Epstein-Barr virus (EBV) in BL cells over 50 years ago led to extensive sero-epidemiology studies and revealed that rather than being a virus restricted to areas where BL is endemic, EBV is ubiquitous in the world's population with an estimated greater than 90% of adults worldwide infected. A second pathogen, Plasmodium falciparum (P. falciparum) malaria is also linked to BL. In this review, we will discuss recent studies that indicate a role for P. falciparum malaria in dysregulating EBV infection, and increasing the risk for BL in children living where P. falciparum malaria transmission is high.

Desde la primera descripción por Denis Burkitt, el linfoma de Burkitt (LB) endémico -el tipo de cáncer pediátrico más común en el África subsahariana- ha guiado a los científicos a investigar este padecimiento en la búsqueda de claves para entender sus orígenes. El descubrimiento desde hace 50 años del virus de Epstein-Barr (VEB) en el LB ha conducido a extensos estudios sero-epidemiológicos y ha revelado que, más que ser un virus restringido a áreas donde el LB es endémico, el VEB es ubicuo en la población mundial, con un estimado mayor del 90% de adultos infectados a escala global. Un segundo agente patógeno se ha ligado al LB, el Plasmodium falciparum (P. falciparum) malaria. En esta revisión se discuten los estudios recientes que indican el papel de P. falciparum malaria en la desregulación de la infección por VEB y en el aumento del riego del LB en niños que viven en regiones con alta transmisión de P. falciparum malaria.