Principle and development of single base editing technology and its application in livestock breeding / 生物工程学报

CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) is widely used in the field of livestock breeding. However, its low efficiency, untargeted cutting and low safety have greatly hampered its use for introducing single base mutations in livestock breeding. Single base editing, as a new gene editing tool, can directly replace bases without introducing double strand breaks. Single base editing shows high efficiency and strong specificity, and provides a simpler and more effective method for precise gene modification in livestock breeding. This paper introduces the principle and development of single base editing technology and its application in livestock breeding.

Transcriptome changes upon Toll-like receptor 9 pathway activation in primary renal tubular epithelial cells / 中华危重病急救医学

Objective:To explore the effect of Toll-like receptor 9 (TLR9) signaling pathway activation on the transcriptome in the renal tubular cells.Methods:Mouse primary renal tubular epithelial cells were extracted and cultured. When the degree of cell fusion reached 80%, they were divided into two groups, which were added with 10 μL phosphate buffered saline (PBS, PBS control group) and TLR9 activator cytosine phosphate guanidine oligodeoxynucleotide (CpG-ODN) with a final concentration of 5 μmol/L (CpG-ODN treatment group). The RNA sequencing was performed on the Illumina platform after extraction. DEGseq software was used to analyze the differential expression of genes between the two groups. Goatools and KOBAS online software were used to analyze the differential genes involved signal pathways. Homer software was used to predict transcription factors.Results:Compared with the PBS control group, there were a total of 584 differentially expressed genes in the CpG-ODN treatment group, of which 102 were up-regulated and 482 were down-regulated. The most significantly enriched gene ontology (GO) terms of differentially expressed genes included response to interferon-β, defense response to virus and other inflammatory pathway. The most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways included 2'-5'-oligoadenylate synthase activity, regulation of ribonuclease activity, negative regulation of virus life cycle, cellular response to interferon-βand defense response to protozoan. The results of transcription factor prediction showed that interferon regulatory factor 3 (IRF3) was the most significantly enriched transcription factor in the promoter sequence of differential genes; the most significant transcription factor downstream of TLR9 was IRF3, and other predicted transcription factors such as transcription factor 21 (TCF21), zinc finger protein 135 (ZNF135), and PR domain containing 4 (PRDM4) might be new candidates for TLR9 signaling pathway.Conclusion:CpG-ODN activates TLR9 signaling pathway, and primary renal tubular epithelial cells can directly respond to CpG-ODN stimulation and undergo transcriptome changes, which provides a basis for further research on the molecular mechanism of TLR9 pathway in sepsis induced acute kidney injury.

CRISPR-mediated base editing in mice using cytosine deaminase base editor 4

BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.

Recent advances and applications of base editing systems / 生物工程学报

The CRISPR system is able to accomplish precise base editing in genomic DNA, but relies on the cellular homology-directed recombination repair pathway and is therefore extremely inefficient. Base editing is a new genome editing technique developed based on the CRISPR/Cas9 system. Two base editors (cytosine base editor and adenine base editor) were developed by fusing catalytically disabled nucleases with different necleobase deaminases. These two base editors are able to perform C>T (G>A) or A>G (T>C) transition without generating DNA double-stranded breaks. The base editing technique has been widely used in gene therapy, animal models construction, precision animal breeding and gene function analysis, providing a powerful tool for basic and applied research. This review summarized the development process, technical advantages, current applications, challenges and perspectives for base editing technique, aiming to help the readers better understand and use the base editing technique.

Magnetic systems for cancer immunotherapy

Immunotherapy is a rapidly developing area of cancer treatment due to its higher specificity and potential for greater efficacy than traditional therapies. Immune cell modulation through the administration of drugs, proteins, and cells can enhance antitumoral responses through pathways that may be otherwise inhibited in the presence of immunosuppressive tumors. Magnetic systems offer several advantages for improving the performance of immunotherapies, including increased spatiotemporal control over transport, release, and dosing of immunomodulatory drugs within the body, resulting in reduced off-target effects and improved efficacy. Compared to alternative methods for stimulating drug release such as light and pH, magnetic systems enable several distinct methods for programming immune responses. First, we discuss how magnetic hyperthermia can stimulate immune cells and trigger thermoresponsive drug release. Second, we summarize how magnetically targeted delivery of drug carriers can increase the accumulation of drugs in target sites. Third, we review how biomaterials can undergo magnetically driven structural changes to enable remote release of encapsulated drugs. Fourth, we describe the use of magnetic particles for targeted interactions with cellular receptors for promoting antitumor activity. Finally, we discuss translational considerations of these systems, such as toxicity, clinical compatibility, and future opportunities for improving cancer treatment.

Detection of free DNA septin 9 gene methylation in plasma / 中南大学学报(医学版)

OBJECTIVES@#To explore the correlation between cytosine-phosphoric-guanylic (CpG) site of Septin 9 gene and colorectal cancer, and to develop a real-time PCR detection system in plasma in patients with colorectal cancer.@*METHODS@#The methylation of training samples was detected by high-throughput sequencing technology, and the sites highly consistent with the clinical information of colorectal cancer were identified. Then the detection system of real-time PCR was designed to analyze the consistency of plasma and tissue based on methylationa sensitive enzyme digestion. Finally, 100 clinical trials were conducted to evaluate the performance of the detection system with the methylation sensitive enzyme digestion-real-time PCR.@*RESULTS@#The highly consistent sites, which were selected by high-throughput sequencing from 71 training set samples, was the 38th CpG. Based on the detection region, the screened methylation sensitive enzymes were @*CONCLUSIONS@#The 38th CpG site of Septin 9 detected by the detection system of methylation sensitive enzyme digestion-real-time PCR can highly predict the occurrence of colorectal cancer with great clinical application value.

TET methylcytosine oxidases: new insights from a decade of research

In primates, males compete for a mate, which is a non-sharable resource. This makes the conditions lessconducive for males to have stable relationships. One such special kind of relationship is a bond where theinteractions are reciprocated, equitable and differentiated. Bonds in macaque societies are based on the degreeof within-group contest competition for mates which is dependent on the synchronization of female fertilephase and reliability of fertility signals. Species of the Fascicularis group, including Nicobar subspecies, showintermediate reliability in the signals with mild peaks, and studies have shown reciprocity but no differentiation. We conducted a study on a group of wild Nicobar long-tailed macaques Macaca fascicularis umbrosusto understand the existing patterns of male-male relationships. We examined whether there is reciprocity inaffiliation among the individuals and whether the rate of affiliation is balanced. We also measured the dominance linearity and steepness in the group to understand the monopolizability of females. We used socialnetwork analysis to understand whether the relations are differentiated based on hierarchical position andwhether the high-ranking individuals are the most central individuals in the distribution of grooming in thegroup. We found that there is reciprocity among the males although that is not equitable. There was no rankrelated differentiation of affiliation among the males of the group. Instead, the identities of individualsinfluenced affiliation patterns. Our results correspond to the existent strong relationships but lack of social bondotherwise found in the Fascicularis group of macaques.

Incorporación de las altas dosis de antraciclina en el tratamiento de la leucemia mieloide aguda del adulto / Incorporation of the high dose of anthracycline in the treatment of adult acute myeloid leukemia

Introducción: La leucemia mieloide aguda representa el 80 por ciento de las leucemias agudas entre los adultos; el tratamiento de inducción a la remisión, para los pacientes menores de 60 años, está basado en la combinación de antraciclinas y arabinósido de citosina. Objetivo: incorporar las altas dosis de antraciclinas al tratamiento de inducción de la leucemia mieloide aguda no promielocítica en pacientes adultos menores de 60 años. Método: Se realizó un estudio cuasiexperimental en 41 pacientes con este diagnóstico, atendidos en el servicio de Adultos del Instituto de Hematología e Inmunología, desde septiembre del 2013 hasta diciembre del 2016. A todos los pacientes se les realizó estudios morfológicos, inmunológicos, citogenéticos y moleculares al inicio de la enfermedad y ecocardiografía para determinar la fracción de eyección y de acortamiento del ventrículo izquierdo al año de finalizado el tratamiento, para determinar la cardiotoxicidad por el uso de las altas dosis de antraciclinas. Resultados: La distribución por edad fue mayor en el grupo de 46 a 52 años representado por el 26,8 por ciento de los casos y predominó el sexo masculino 60,9 por ciento. En el 85 por ciento de los casos la enfermedad apareció de novo. Según los criterios morfológicos de la clasificación Franco Británico Americana el 31,7 por ciento correspondió a la variante M1, en estrecha relación con las determinaciones por citometría de flujo, para esta variedad. Los genes más comúnmente involucrados fueron el NPM1 y el AML/ETO, para el 24 por ciento y 22 por ciento, respectivamente. El 56,1 por ciento de los pacientes alcanzó la remisión hematológica con un solo ciclo de tratamiento y el 14,6 por ciento, necesitó realizar un segundo esquema de inducción. No se reportaron eventos de cardiotoxocidad por antraciclina durante el tratamiento, ni al año de culminado este. Conclusiones: Con el uso de las altas dosis de antraciclina se lograron remisiones hematológicas, sin toxicidad cardiovascular demostrada(AU)

Introduction: Acute myeloid leukemia represents 80 percent of acute leukemias among adults; the induction treatment to obtain remission in patients under 60 years old is based on the combination of anthracyclines and cytosine arabinoside. Objective: to incorporate the high doses of anthracyclines to the treatment of induction of non-promyelocytic acute myeloid leukemia in adult patients under 60 years of age. Method: We conducted a quasi-experimental study in 41 patients with this diagnosis, at the adult clinic service of the Institute of Hematology and Immunology, from september 2013 to december 2016. Morphological, immunological, cytogenetic and molecular studies were carried out at the beginning of the disease and also echocardiography was performed to determine the ejection fraction and shortening of the left ventricle a year after the end of treatment, to determine cardiotoxicity due to the use of high doses of anthracyclines. Results: The distribution by age was higher in the group of 46 to 52 years represented by 26.8 percent of the cases and the male sex predominated 60.9 percent. In 85 percent of the cases the disease appeared de novo. According to the morphological criteria of the French American British classification, 31.7 percent corresponded to the M1 variant, in close relation with the determinations by flow cytometry, for this variety. The genes most commonly involved were NPM1 and AML / ETO, for 24 percent and 22 percent respectively. 56.1 percent of patients achieved hematological remission with a single treatment cycle and 14.6 percent of patients needed a second induction scheme. No anthracycline cardiotoxicity events were reported during the treatment, nor a year after the treatment, in the patients evaluated. Conclusions: With the use of high doses of anthracycline, have been hematological remissions, without demonstrated cardiovascular toxicity(AU)

Qualitative and quantitative determination of Sophora flavescens seeds and analysis of its blood components based on UHPLC-MS/MS / 中草药

Objective Based on UPLC-Q-Exactive orbitrap high-resolution mass spectrometry, qualitative analysis of Sophora flavescens seeds and its blood components were analyzed, and UHPLC-MS/MS quantitative analysis of six alkaloids in Sophora flavescens seeds were performed. Methods The separation was performed on the Waters HSS T3 column (100 mm × 2.1 mm, 1.7 μm), with mobile phase 0.1% formic acid acetonitrile (10 mmol/L ammonium acetate) solution (A) and 0.1% formic acid water (10 mmol/L ammonium acetate) solution (B) for gradient elution. Electrospray (ESI) fog ion source was used in mass spectrometry; Data were collected in positive ion mode for qualitative and quantitative analysis. Results A total of 34 chromatographic peaks were identified in the samples of Sophora flavescens seeds. Most of them were 26 alkaloids. A total of 18 alkaloids were identified in the plasma samples. The content of six alkaloids in Sophora flavescens seeds was up to 18.5%, and the content of oxymatrine was the highest. Conclusion The acute toxity of Sophora flavescens seeds may be due to the alkaloids entering into blood directly. This study provides a reference for the development and utilization of Sophora flavescens seeds.

A Potential Therapy Using Engineered Stem Cells Prevented Malignant Melanoma in Cellular and Xenograft Mouse Models / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: In the present study, human neural stem cells (hNSCs) with tumor-tropic behavior were used as drug delivery vehicle to selectively target melanoma. A hNSC line (HB1.F3) was transduced into two types: one expressed only the cytosine deaminase (CD) gene (HB1.F3. CD) and the other expressed both CD and human interferon-β (IFN-β) genes (HB1.F3.CD. IFN-β). MATERIALS AND METHODS: This study verified the tumor-tropic migratory competence of engineered hNSCs on melanoma (A375SM) using a modified Boyden chamber assay in vitro and CM-DiI staining in vivo. The antitumor effect of HB1.F3.CD and HB1.F3.CD.IFN-β on melanoma was also confirmed using an MTT assay in vitro and xenograft mouse models. RESULTS: A secreted form of IFN-β from the HB1.F3.CD.IFN-β cells modified the epithelial-mesenchymal transition (EMT) process and metastasis of melanoma. 5-Fluorouracil treatment also accelerated the expression of the pro-apoptotic protein BAX and decelerated the expression of the anti-apoptotic protein Bcl-xL on melanoma cell line. CONCLUSION: Our results illustrate that engineered hNSCs prevented malignant melanoma cells from proliferating in the presence of the prodrug, and the form that secreted IFN-β intervened in the EMT process and melanoma metastasis. Hence, neural stem cell-directed enzyme/prodrug therapy is a plausible treatment for malignant melanoma.

Comparison of the phenolic contents and epigenetic and genetic variability of wild and cultivated watercress (Rorippa nasturtium var. aquaticum L.)

Background: Epigenetic modifications are key factors modulating the expression of genes involved in the synthesis of phytochemicals. The knowledge of plant epigenetic and genetic variations can contribute to enhance the production of bioactive compounds. These issues have been little explored thus far in Rorippa nasturtium var. aquaticum L. (watercress), an edible and medicinal plant. The aim of the current study was to determine and compare the phenolic composition and epigenetic and genetic variations between wild and cultivated watercress. Results: Significant differences were found in the quantitative phenolic composition between wild and cultivated watercress. The eight primer combinations used in the methylation-sensitive amplification polymorphism (MSAP) method revealed different epigenetic status for each watercress type, the cultivated one being the most epigenetically variable. The genetic variability revealed by the EcoRI/MspI amplification profile and also by eight inter-simple sequence repeat (ISSR) primers was different between the two types of watercress. The results of the Mantel test showed that the correlation between genetic and epigenetic variations has diminished in the cultivated type. Cluster analyses showed that the epigenetic and genetic characterizations clearly discriminated between wild and cultivated watercress. Conclusions: Relevant chemical, epigenetic, and genetic differences have emerged between wild and cultivated watercress. These differences can contribute to fingerprint and develop quality control tools for the integral and safety use and the commercialization of watercress. The richness of epialleles could support the development of tools to manipulate the watercress epigenome to develop high bioproduct­producing cultivars

Expression of cytidine triphosphate synthetase 1 in pancreatic cancer cell lines and its clinical significance / 中华普通外科杂志

Objective To investigate the cytidine triphosphate synthetase 1 (CTPS1) expression in pancreatic cancer cell lines and its impaction on prognosis.Methods The GEO profiles database was searched to collect clinical data of 39 patients of pancreatic ductal adenocarcinoma.Kaplan-Meier curve was used to analyze the relationship between the patients' prognosis and the expression of CTPS1.qPCR and Western blot were applied to detect the expression of CTPS1 in 4 pancreatic cancer cell lines.Results The mRNA expression of CTPS1 in Sw1990,BxPC-3,Panc1,and MIA-Capa2 was 1.00) ± 0.20,2.92±0.95,4.29±0.14,and 2.26±0.33 (t=-16.19,-11.45,-8.09,-13.12,P＜0.05) when compared to the normal pancreatic ductal epithelial cell line HPDE of 7.70 ± 0.72.The relative protein expression of CTPS1 in pancreatic cancer cell lines Sw1990,BxPC-3,Panc1,and MIA-Capa2 was 0.40 ± 0.06,0.20±0.09,0.68±0.11,and0.48±0.06 (t=-8.97,-10.5,-4.39,-7.88,P＜0.05) when compared to HPDE of 1.09 ±0.12.The overall survival (OS) in patients with higher CTPS1 expression than adjacent tissue was 28 months (95％ CI:9.2-46.8 months),and that with lower CTPS1 expression was 13 months (95％ CI:8.2-17.8 month),(x2 =4.02,P＜0.05).Conclusions CTPS1 in the pancreatic cancer cell lines are of low expression,and the level is positively related to patients' survival time.

El caseinato de sodio incrementa número de linfocitos B en ratones / Sodium caseinate increases the number of B lymphocytes in mouse

Introducción. El caseinato de sodio, una sal de la caseína utilizada como agente proinflamatorio en ratones, es capaz de inducir granulopoyesis en vivo e incrementar la producción de citocinas esenciales en dicho evento. Objetivo. Evaluar si el caseinato de sodio es capaz de inducir un efecto biológico en células de origen linfoide y la producción de citocinas involucradas con este linaje. Materiales y métodos: Se utilizaron ratones hembra BALB/c de 8 a 12 semanas de edad. Los animales se inyectaron cuatro veces, con intervalos de 48 horas, por vía intraperitoneal con 1 ml de caseinato de sodio (10 % de SFB p/v). La población de linfocitos B y la incorporación de bromodesoxiuridina (BrdU) se analizaron mediante citometría de flujo. La detección de la interleucina 7 se evaluó mediante la técnica de ELISA. Resultados. Tras la inyección por vía intraperitoneal, el número de linfocitos B 220+ provenientes del bazo de ratones tratados con caseinato de sodio aumentó comparados con los que solo recibieron el vehículo como tratamiento (89,01±1,03 Vs. 75,66±2,08), así como la incorporación de BrdU en células B220+ (38,59±4,48 Vs. 11,82±1,04). Se evidenció, asimismo, el incremento en la concentración de la interleucina 7 (IL-7) en el suero de los ratones tratados con caseinato de sodio, comparados con los que solo recibieron el vehículo (62,1±17,5 Vs. 26,9±4,4 pg/ml). Conclusión. El caseinato de sodio fue capaz de aumentar el número de linfocitos B en bazo de ratones, así como inducir la producción de IL-7, citocina clave para la linfopoyesis B.

Introduction: Sodium caseinate, a casein salt, is a proinflammatory agent in mice, and it is able to induce granulopoiesis in vivo and to increase the production of cytokines, which is key for this biological process. Objective: To assess whether sodium caseinate is able to induce a biological effect on cells from lymphoid origin and the production of cytokines involved in this lineage in vivo. Materials and methods: We used female BALB /c mice from 8 to 12 weeks old. The animals were injected intraperitoneally (IP) with 1 ml of sodium caseinate (10% PBS w/v) four times every 48 hours. The B cell populations and the incorporation of BrdU were analyzed by flow cytometry. Detection of interleukin-7 was assessed by ELISA (Enzyme-Linked ImmunoSorbent Assay). Results: We established that after intraperitoneal injection, the number of B lymphocytes 220+ from the spleen of mice treated with sodium caseinate increased compared to those that only received the vehicle (89.01±1.03 vs 75.66 ± 2.08), and the same was observed with the incorporation of BrdU in B220 + cells (38.59±4.48 vs 11.82±1.04 respectively). We also established that the concentration of interleukin-7 (IL-7) in the serum of mice treated with sodium caseinate increased compared to those that only received the vehicle (62.1 ± 17.5 vs 26.9 ± 4.4 pg/ml). Conclusion: Sodium caseinate was able to increase the number of B lymphocytes in the spleen; it also induced IL-7 production, a cytokine that is key for the B cell lymphopoiesis.

Apoptosis induced by transferring cytosine deaminase suicide gene into kinds of cancer cells with novel polyamidoamine dendrimers / 药学学报

This study demonstrates efficacy of a novel polyamidoamine dendrimers (PAMAM dendrimers) with pentaerythritol derivatives as the core (G5 PD dendrimer) in deliver of the cytosine deaminase (CD) gene and EGFP gene fusion plasmid into different tumor cell lines to induce apoptosis. The physical and chemical properties of G5 PD dendrimers in terms of DNA complexation, particulate properties and transfection efficiencies were investigated and compared with commercial gene vectors PEI 25 kDa. The optimum ratio of G5 PD dendrimer complexed with plasmid DNA was 0.2:1, and the particle size of the complex was (100±5) nm. Compared with the commercial gene carriers PEI, G5 PD dendrimer exhibited a higher transfection efficiency at the weight ratio of 1:1 in three different cell lines, given the fact that PEI are different from PAMAM dendrimers in terms of molecular structure. Furthermore, the cytotoxicity assays of the cell lines transfected with G5 PD dendrimer/pCD-EGFP-N1 followed by exposure to various concentrations of 5-fluorocytosine (5-FC) also showed that the transfected cell lines could generate a very low amount of 5-FC to 5-fluorouracil (5-FU) in a short period of time, which indicating the high expression level of CD gene in the cell line. These results indicate that the CD/5FC system of G5 PD dendrimer has an excellent efficacy in gene delivery.

Determination of Global DNA Methylation Levels in Medicinal Plants by LC-MS/MS / 中国药房

OBJECTIVE:To develop a detection LC-MS/MS method for global DNA methylation in medicinal plants. METH-ODS:Genomic DNA was isolated using plant DNA extraction kit,and then hydrolyzed by 88% formic acid at 140 ℃. After dried with nitrogen,extracted DNA was dissolved again with mobile phase. LC separation was performed on HILIC column with mobile phase consisted of 7 mmol/L ammonium formate-acetonitrile (gradient elution) at flow rate of 0.3 ml/min. The analysis was con-ducted by tandem MS with positive ion electrospray ionization in multiple reaction monitoring(MRM)mode. The ratio of genomic DNA methylation in 10 commonly used medicinal plants was calculated. RESULTS:The linear ranges of Cyt and 5mC were 1-500 ng/ml(r=0.999 5)and 0.2-100 ng/ml(r=0.999 6). The relative standard deviations(RSDs)of accuracy were 1.12% and 3.68%(n=6). The RSDs of intra-day precision were 2.36% and 4.02% for Cyt and 5mC,respectively (n=5). The RSDs of inter-day precision were 1.04% and 3.54% for Cyt and 5mC,respectively (n=3). The RSDs of repeatability test were 1.53% and 3.27%for Cyt and 5mC,respectively(n=6). The recoveries of Cyt and 5mC were 98.7%-102.1% and 91.2%-103.5%. The percentages of global DNA methylation in 10 medicinal plants were ranged from 17.63% to 25.18%. CONCLUSIONS:LC-MS/MS method is simple,rapid,sensitive and precise,and can be used for the detection of global DNA methylation in medicinal plants.

Determination of nine kinds of nucleosides in Spirodelae Herba from different habitats by UPLC-MS/MS / 中草药

Objective: To establish a UPLC-MS/MS method for the simultaneous determination of nine kinds of nucleosides in Spirodelae Herba, and analyze and investigate the difference of nucleosides ingredients in samples from 13 habitats. Methods: The determination was performed on the Waters XbridgeTM Amide-C18 (150 mm × 4.6 mm, 3.5 μm), the 0.1% formic acid-water (A) and 0.1% acetonitrile-water (B) in gradient elution as mobile phase. The flow rate was 0.5 mL/min. The column temperature was 30 ℃. MS instrument was equipped with ESI+ ion source. Gaining the extracted ion chromatograms, then the peak area for quantitative, principal component analysis (PCA), and hierarchical clustering analysis (HCA) were used to comprehensive evaluation. Results: All nine kinds of nucleoside showed good linearity (r ≥ 0.998 9). The RSD of the precision, repeatability, and stability tests were less than 3.5%. The average recovery rates were in the range of 94.4%-101.9%, RSD were in the range of 1.73% - 3.58%. There are differences in the composition and content from different habitats. The content of 2'-deoxyuridine and 2'-deoxyinosine in the lower levels, cytosine was the lowest, while the contents of uridine, inosine, and xanthine were in higher levels. The largest content was in Spirodelae Herba from Huangshan, Anhui province. Conclusion: The method for the simultaneous determination of the nine kinds of nucleosides is simple, accurate, and reproducible, that will provide the basis for the formulation of herbs Spirodelae Herba for quality control standards.

Research progress on molecular mechanism of sporadic colorectal cancer / 实用肿瘤学杂志

Colorectal cancer( CRC) is one of the most common tumor,which has complicated pathogene-sis.it is estimated that the vast majority of CRCs is non-hereditarysporadic cancerswith no apparent evidence of an inherited component.Sporadic CRC results from the cumulative effects of multiple genetic and epigenetic al-terations caused by somatic mutations, which may be the indirect result of several environmental factors them-selves.There are at least 3 major genetic alternations that lead to colorectal carcinogenesis:(1)The chromosomal instability(CIN)pathway;(2)The microsatellite instability(MSI)pathway;(3)The cytosine-phospho-guanine ( CpG) island methylator phenotype( CIMP) pathway,while DNA methylation,modifications in histone proteins and microRNAs( miRNAs) are analyzed in the field of epigenetic alterations.This review summarizes the newest bio-molecular progression involved in CRC pathogenesis,for the purpose of improving strategy for prevention,surveil-lance,early diagnosis and therapy.

Tetrandrine affects the expression of DNMT in HL-60 cells / 中国医师杂志

Objective To investigate the expression of DNA methyltransferase (DNMT) in HL-60 cells induced by tetrandrine (Tet).Methods HL-60 cells were treated with different concentrations of Tet and decitabine (DAC) alone and in combination with both.Methyl thiazolyl tetrazolium (MTT) assay was used to assess cytotoxic effect.Flow cytometry (FCM) was used to determine apoptosis rate.Real-time quantitative polymerase chain reaction (PCR) assay was used to quantify mRNA levels of DNMT.Western blot was used to quantify the expression of DNMT protein.Results Tet inhibited the growth and proliferation of HL-60 cells in a time-and dose-dependent manners (both P ＜0.01).Tet treated HL-60 cells after 48 h at the concentration of 2 μmol/L,and 4 μmol/L,the levels of DNMT gene and protein in the drug administration group decreased compared to the control group.After incubation for 48 h with Tet 2 μmol/L combined with DAC 4 μmol/L,the combination group was significantly depressed.Conclusions Tet could potently inhibit the growth and proliferation of HL-60 cells,reduce the expression levels of DNMT mRNA and protein,and have a more obvious effect in the combination group.

Influence of the cytosine content in liposome-coated 99Tcm-HYNIC-survivin ASODN on tumor bearing nude mice imaging / 中华核医学与分子影像杂志

Objective To investigate whether specific cellular uptake of 99Tcm-survivin-ASODN in nude mice bearing human HCC is influenced by its cytosine content.Methods Three kinds(A1,A2,A3) of synthesized survivin ASODN with three cytosine contents(10％,20％,30％),20 bases per single-strand were prepared.They were labeled with 99Tcm by conjugating with a bifunctional chelator HYNIC,purified through Cellufine GH-25 and then encapsulated with liposome.Antisense gene imaging and the biodistribution of 99Tcm-HYNIC-survivin ASODN in nude mice bearing HCC were performed.The data were analysed by Kruskal-Wallis H test.Results At 4 h post injection,all the 3 labelled compounds showed increased uptake by tumor,liver and kidney.With increase in cytosine content,the uptake increased in kidney (％ID/g:1.50±0.06,2.80±0.09 and 3.96±0.03),and decreased in tumor (％ID/g:2.08±0.08,1.69±0.01 and 1.20±0.09).T/NT in imaging (4.49-4.93,4.12-4.21,3.35-3.85;H=12.50,P＜0.05) and in biodistribution (4.08-4.94,4.02-4.18,3.66-3.85;H=10.82,P＜0.05) were all significantly different.Conclusion ASODN with lower cytosine content shows higher uptake by HCC tumor cells and less stasis in kidneys,thus providing better quality in antisense gene imaging.

Medulative effect of dihydroartemisinin on UCHL1 gene expression in human prostate cancer PC-3 cells and its mechanism / 肿瘤

Objective: To investigate the effect of dihydroartemisinin (DHA) on expression of tumor suppressor gene ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in human prostate cancer cell line PC-3, and explore its regulative mechanism. Methods: PC-3 cells were treated with different concentrations (25, 50, and 100 μmol/L) of DHA for 48 h, while PC-3 cells without DHA treatment were used as the control. Then the apoptosis and cell cycle distribution were detected by flow cytometry. The expressions and cellular locations of DNA methyltransferase 1 (DNMT1) and UCHL1 proteins were detected by immunofluorescence staining. The expression levels of UCHL1, DNMT1, phospho-Akt (p-Akt) and Akt proteins were detected by Western blotting. The 1 μmol/L phosphoinositide-3-kinase (PI3K) inhibitor Wortmanin was used as a positive control to analyze the expression of p-Akt protein in DHA-treated group. Results: DHA significantly induced the apoptosis of PC-3 cells and arrested the cell cycle distribution at phase G2/M as compared with those of the control group (both P 0.05) in the DHA-treated group and the positive control group. The downregulation of p-Akt expression was more obvious in high-concentration of DHA-treated group, much closer to that in the positive control group. Conclusion: DHA can inhibit the expression of DNMT1, restore the function of UCHL1 gene, induce the apoptosis of PC-3 cells, and block the cell cycle progression. These mechanisms may be related to the suppressive activity of PI3K/Akt pathway.