In Vivo Improvements in Facial Appearance and in Vitro Changes in Gene Expression Using a Topical Formulation Designed to Repair Environmentally Induced DNA Damage

Background: While sunscreen has been accepted as a mainline defence against photodamage from ultraviolet, visible light and near-infrared radiation, there appears to be a lack of research into photorepair. The concept of protecting the skin during the day and repairing cellular damage at night is intuitive, yet specific strategies revolving around combinations of proven reparative active ingredients remain unelucidated. Purpose: To investigate the efficacy of a solar repair Formulation following ultraviolet and environmental exposure in order to improve overall skin health and appearance through three hypotheses: The Formulation increases expression of DNA repair mechanisms markers;The Formulation enhances overall skin appearance through reducing signs of inflammation, elevating hydration, reinforcing skin firmness and amplifying radiance;In-Vivo efficacy test results are aligned with measured gene expression changes. Methods: The Formulation (#6NIC1.V1.1-1) was tested for: In-vitro LDH cytotoxicity activity, In-vitro qPCR gene expression with and without ultraviolet exposure on a reconstructed 3-dimensional skin model, and In-Vivo efficacy study on a panel of 22 participants objectively and subjectively. Results: Skin radiance, firmness, hydration, redness, and inflammation are significantly improved after In-Vivo skin exposure to the Formulation and environmental challenges such as ultraviolet radiation. These outcomes were confirmed by in-vitro genetic testing on a reconstructed human skin model. Conclusion: The studies allowed us to identify and group results in four main skin functions that were significantly enhanced following the application of the Formulation: firmness, hydration, radiance and soothing.

Cloning of M and NP Gene of H5N1 Avian Influenza Virus and Immune Efficacy of their DNA Vaccines

H5N1 鸟的流行性感冒病毒(A/chicken/Hubei/489/2004 ) 的 M 和 NP 基因被 RT-PCR 从病毒的 RNA 放大，并且分别地克隆向量进 pMD18-T。包含 M 基因(pHM6-m ) 或 NP 基因(pHM6-np ) 的表示 plasmid 然后被把 M 或 NP 基因插入到 pHM6 优核质表示向量构造；构造 plasmid 然后被定序。32 只 BALB/c 老鼠(6-week-old ) 在随机被划分成四个组。三组 BALB/c 老鼠被接种一次有 plasmid pHM6-m， plasmid pHM6-np 的 30 渭 g 或 plasmid pHM6-m (15 渭 g ) 和 pHM6-np (15 渭 g ) 的混合的任何一个 30 渭 g 的肌内的线路分别地。老鼠的一个另外的组作为控制与 100 渭 l PBS 被注射。二个星期以后，所有老鼠与相应 H5N1 鸟的流行性感冒病毒被质问，并且在下列 12 天内观察了。在 pHM6-m 组， pHM6-np 组和混合 plasmids 组的老鼠的幸存率分别地是 62.5% ， 25.0% 和 50.0% 。结果证明有效保护能被 pHM6-m 或 pHM6-np 提供，但是 pHM6-m 比 pHM6-np 提供了更好保护的效果。关键词 H5N1 流行性感冒病毒 - M 基因 - NP 基因 - 克隆 - DNA 疫苗的 CLC 数字 S852.65 基础条款：国家基本科学才能训练资助(NSFC J0630648 )

Mitochondrial DNA Markers for PCR-Based Phylogenetic Analysis of Ark Shells

Arcidae species are commercially important bivalves in Japan and are commonly referred to as bloody ark due to their red blood. They have thick shells with distinct radiating ribs, and the numbers of these ribs are important morphological features for species discrimination. However, some Arcidae species are morphologically indistinguishable, with a similar number of the ribs in adults and deficient rib formation, particularly among juveniles. Thus, we developed a reliable molecular marker to genetically discriminate between 7 Arcidae species belonging to Scapharca, Anadara, and Tegillarca based on species-specific polymorphic segments of mitochondrial DNA. PCR amplification of partial COI, 16S rRNA, 12S rRNA, and Cyt b genes was performed on 7 species using 8 primer sets. Only the set of Scapharca-specific forward primer and universal reverse primer for the partial COI gene successfully yielded single PCR products from all 7 species examined. Thus, nucleotide sequences of 481 bp portion of these PCR products were determined, and the degrees of nucleotide substitutions ranged from 0.4% between S. broughtonii and T. granosa to 20.2% between S. satowi and A. antiquata. In addition, a phylogenetic tree showed significant differences between 7 species, with higher bootstrap support than 69.

Promoter Hypermethylation of DNA Repair Gene MGMT in Laryngeal Squamous Cell Carcinoma

The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, tissues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 （34.8 %） laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades （χ^2= 3. 130, P=0. 077） or in samples from patients with different TNM status （χ^2= 3. 957, P=0. 138）. No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.

The cloning of 3'-truncated preS/S gene from HBV genomic DNA and its expression in transgenic mice

INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes

Cloning of Bt cry Genes by Rapid Screening of DNA Libraries with PCR-RFLP

Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, crylCa insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR I fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR I complete and 5au3A I partial digestion. On the basis of every 50 transformants pooled together from 5-10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa , and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletropora-tion. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.

A DNA delivery system containing listeriolys in Oresults in enhanced hepatocytedirected gene expression

AIM To determine whether incorporation of the pH dependent bacterial toxin listeriolysin O (LLO) into the DNA carrier system could increase the endosomal escape of internalized DNA and result gene expression. METHODS A multi component delivery system was prepared consisting of asialoglycoprotein (ASG), poly L lysine (PL), and LLO. Two marker genes, luciferase and β galactosidase in plasmids were complexed and administered in vitro to Huh7[ASG receptor (+) ] and SK Hep1[ASG receptor (-) ] cells. Purity, hemolytic activity, gene expression, specificity, and toxicity were evaluated. RESULTS An LLO containing conjugate retained cell targeting specificity and membranolytic activity. In ASG receptor (+) cells, luciferase gene expression was enhanced by more than 7 fold over that of conjugates without the incorporation of listeriolysin O. No significant expression occurred in ASG receptor (-) cells. Enhancement of β galactosidase gene expression was less, but still significantly increased over controls. There was no detectable toxicity at concentrations shown to be effective in transfection studies. CONCLUSIONS ASOR PL can be coupled to LLO using disulfide bonds, and successfully target and increase the gene expression of foreign DNA.

Studies on DNA Vaccine of the Conservative Region of Gene Encoding the Male-Specific Gynecophoral Canal Protein of Schistosoma japonicum (Chinese strain)

In order to observe the protective effect induced by vaccinating animals with the DNA vaccine of Sex-specific expression gene of Schistosoma, A 868 bp cDNA fragment amplified by RT-PCR from adult Schistosoma japonicum (Chinese strain) mRNA was cloned into the eukaryotic expression vector pcDNA3 and the recombinant eukaryotic expression vector pcDNA3-SjGCP1 was directly injected into BALB/c mice intramuscularly 3 times with the interval of 3 weeks .Both the vaccinated and control group of mice were challenged with 40 cercariae of Sj 5 weeks after last injection and perfused 7 weeks post-challenge. The worm and egg reduction rate got from vaccinated mice was 32.4% and 46.9% respectively. The result indicated that pcDNA3-SjGCP1 DNA vaccine induces the significant protection in animal against Schistosoma japonicum infection.

Amplification and function analysis of N6-adenine-specific DNA methyltransferase gene in Nilaparvata lugens

Methylation of the N6 position of adenine, termed N6-methyladenine, protects DNA from restriction endonucleases via the host-specific restriction-modification system. N6-methyladenine was discovered and has been well studied in bacteria. N6-adenine-specific DNA methyltransferase（N6AMT） is the main enzyme catalyzing the methylation of the adenine base and knowledge of this enzyme was mainly derived from work in prokaryotic models. However, large-scale gene discovery at the genome level in many model organisms indicated that the N6AMT gene also exists in eukaryotes, such as humans, mice, fruit flies and plants. Here, we cloned a N6AMT gene from Nilaparvata lugens（Nlu-N6AMT） and amplified its fulllength transcript. Then, we carried out a systematic investigation of N6AMT in 33 publically available insect genomes, indicating that all studied insects had N6AMT. Genomic structure analysis showed that insect N6AMT has short introns compared with the mammalian homologs. Domain and phylogenetic analysis indicated that insect N6AMT had a conserved N6-adenine Mlase domain that is specific to catalyze the adenine methylation. Nlu-N6AMT was highly expressed in the adult female. We knocked down Nlu-N6AMT by feeding ds RNA from the second instar nymph to adult female, inducing retard development of adult female. In all, we provide the first genome-wide analysis of N6AMT in insects and presented the experimental evidence that N6AMT might have important functions in reproductive development and ovary maturation.

Negative effects of long-term feeding of high-grain diets to lactating goats on milk fat production and composition by regulating gene expression and DNA methylation in the mammary gland

Background： It is well known that feeding a high concentrate（HC） diet to lactating ruminants likely induces subacute ruminal acidosis（SARA） and leads to a decrease in milk fat production. However, the effects of feeding a HC diet for long periods on milk fatty acids composition and the mechanism behind the decline of milk fat still remains poorly understood. The aim of this study was to investigate the impact of feeding a HC diet to lactating dairy goats on milk fat yield and fatty acids composition with an emphasis on the mechanisms underlying the milk fat depression. Seventeen mid-lactating dairy goats were randomly allocated to three groups. The control treatment was fed a low-concentrate diet（35% concentrate, n = 5, LC） and there were two high-concentrate treatments（65% concentrate, HC）, one fed a high concentrate diet for a long period（19 wks, n = 7, HL）; one fed a high concentrate diet for a short period of time（4 wk, n = 5, HS）. Milk fat production and fatty acids profiles were measured. In order to investigate the mechanisms underlying the changes in milk fat production and composition,the gene expression involved in lipid metabolism and DNA methylation in the mammary gland were also analyzed.Results： Milk production was increased by feeding the HC diet in the HS and HL groups compared with the LC diet（P 〈 0.01）, while the percentage of milk fat was lower in the HL（P 〈 0.05） but not in the HS group. The total amount of saturated fatty acids（SFA） in the milk was not changed by feeding the HC diet, whereas the levels of unsaturated fatty acids（UFA） and monounsaturated fatty acids（MUFA） were markedly decreased in the HL group compared with the LC group（P 〈 0.05）. Among these fatty acids, the concentrations of C15：0（P 〈 0.01）, C17：0（P 〈 0.01）, C17：1（P 〈 0.01）, C18：1 n-9 c（P 〈 0.05）, C18：3 n-3 r（P 〈 0.01） and C20：0（P 〈 0.01） were markedly lower in the HL group, and the concentrations of C20：0（P 〈 0.05） and C18：3 n-3 r（P 〈 0.01） were lower in the HS group compared with the LC group. However, the concentrations of C18：2 n-6 c（P 〈 0.05） and C20：4 n-6（P 〈 0.05） in the milk fat were higher in the HS group. Real-time PCR results showed that the m RNA expression of the genes involved in milk fat production in the mammary gland was generally decreased in the HL and HS groups compared with the LC group. Among these genes, ACSL1, ACSS1 ＆ 2, ACACA, FAS, SCD, FADS2, and SREBP1 were downregulated in the mammary gland of the HL group（P 〈 0.05）, and the expressions of ACSS2, ACACA, and FADS2 m RNA were markedly decreased in the HS goats compared with the LC group（P 〈 0.05）. In contrast to the gene expression, the level of DNA methylation in the promoter regions of the ACACA and SCD genes was increased in the HL group compared with the LC group（P 〈 0.05）. The levels of ACSL1 protein expression and FAS enzyme activity were also decreased in the mammary gland of the HL compared with the LC group（P 〈 0.05）.Conclusions： Long-term feeding of a HC diet to lactating goats induced milk fat depression and FAs profile shift with lower MUFAs but higher SFAs. A general down-regulation of the gene expression involved in the milk fat production and a higher DNA methylation in the mammary gland may contribute to the decrease in milk fat production in goats fed a HC diet for long time periods.

Detection of fusion gene in cell-free DNA of a gastric synovial sarcoma

Synovial sarcoma(SS) is genetically characterized by chromosomal translocation, which generates SYT-SSX fusion transcripts. Although SS can occur in any body part, primary gastric SS is substantially rare. Here we describe a detection of the fusion gene sequence of gastric SS in plasma cell-free DNA(cf DNA). A gastric submucosal tumor was detected in the stomach of a 27-year-old woman and diagnosed as SS. Candidate intronic primers were designed to detect the intronic fusion breakpoint and this fusion sequence was confirmed in intron 10 of SYT and intron 5 of SSX2 by genomic polymerase chain reaction(PCR) and direct sequencing. A locked nucleic acid(LNA) probe specificto the fusion sequence was designed for detecting the fusion sequence in plasma and the fusion sequence was detected in preoperative plasma cfD NA, while not detected in postoperative plasma cfD NA. This technique will be useful for monitoring translocation-derived diseases such as SS.

Sequencing and Analysis of G HR Gene from Genomic DNA Pools of Mink

In order to study the single nucleotide polymorphisms （SNPs） of growth hormone receptor （GHR） gene in mink populations, genomic DNA pools of Minghua black minks and silver-blue minks were constructed, and the 10^th exon of GHR gene was PCR amplified from the two DNA pools and sequenced. The results showed that two SNPs were found at position 209 （T/C） and position 533 （C/A） of the 10^th exon of GHR gene in the two mink populations.

GENE DIAGNOSIS OF HEMOGLOBINOPATHY IN CHINESE BY AMPLIFIED DNA

This paper describes the application of DNA amplification in vitro with the polymerase chain reaction on the prenatal diagnosis of thalassemia syndromes and on detection of HbS and HbD Punjab genes. DNA polymerase chain reaction was performed on lysed amniotic fluid cells or chorionic villus samples without prior DNA extraction and on DNA sampling from dried blood spots on filter paper blotters, α-thalassemia was prenatally diagnosed by direct analysis of amplified fetal target sequences on gel electrophoresis; and β-thalassemia was predicted by Hgi AI RFLP linkage analysis with amplified β-globin DNA. HbS and HbD Punjab genes were identified by Mst Ⅱor Eco RI mapping of the amplified β-globin DNA directly on the electrophoretic gels. The analysis of the amplified DNA does not require radioactive DNA probes and Southern hybridization. The total procedure can be completed within five hours.

A New Tandem Gene Construction Method Involving a Cloning System Using <i>Poxvirus DNA polymerase</i>, and Its Application to Gene Expression

A simple method for constructing polymerized genes using only restriction enzymes and commercially available cloning systems was established. In this system, gel isolations or purifications of target genes after restriction enzyme digestions or PCR amplifications, which often cause errors and mutations in the target gene sequence, are not necessary. To verify the usefulness of this method, one, two, four, eight, and sixteen tandem-repeats of the Green Fluorescent Protein (GFP) expression gene in Escherichia coli were sequentially constructed. Efficacies of the GFP gene expression of those plasmids in E. coli showed an increasing trend in accordance with the copy numbers of the gene. On SDS polyacrylamide gel electrophoresis with Coomassie blue staining, no expressed protein could be seen in E. coli cells harboring plasmids that contained one or two copies of the gene. However, expressed protein bands in E. coli cells were clearly detected with 4 copies of the gene. In quantitative analyses involving green fluorescence intensities per culture volume, the expression level in E. coli with 16 copies of the gene was 36.3-fold higher than that in E. coli with one copy at 22 hours after induction.

Association between the DNA Repair Gene Polymorphisms and Lung Cancer in Turkish Population

Introduction: DNA repair enzymes continuously monitor DNA to correct damaged nucleotide residues generated by exposure to environmental mutagenic and cytotoxic compounds or carcinogens. Our objective was to investigate the association among XRCC1 (Arg399Gln and Arg194Trp), XRCC3 (Thr241Met), XPD-ERCC2 (Lys751Gln), APE1 (Asp241Glu), PARP-ADPRT (Val762Ala) DNA repair gene polymorphisms and lung cancer in Turkish population. Materials and Methods: Our patient group consists of 90 patients with lung cancer and the control group had 100 healthy individuals all of those smoking. DNA was extracted using the whole blood samples. PCR- RFLP technique was used to investigate the polymorphisms on target genes. Results: There was no significant difference in the genotype distributions of XPD Lys751Gln, XRCC1 Arg194Trp, XRCC3 Thr241Met, APE1 Asp241Glu between lung cancer patients and controls for each polymorphism (p > 0.05). However, there was a significant difference between the genotype distributions of XRCC1 Arg399Gln, and PARP Val762Ala in patients and the control group (p > 0.05). Discussion: Only the polymorphisms of XRCC1 codon 399 and PARP Val762Ala alleles are associated with the risk of lung cancer. Other genotypes were not related to lung cancer.

Molecular Characterization and Phylogenetic Analysis of Culex quinquefasciatus by DNA Barcoding

The southern house mosquito, Culex quinquefasciatus, predominantly exists all through the tropics acts as a vector causing several parasitic diseases;it is very important mosquito in transmitting certain diseases which include filariasis. The progression of this disease is due to swift urbanization. In fact, morphological identification mosquito species is very difficult task;moreover, origin of the species is unpredictable. Recently, DNA barcoding of partial mitochondrial COI gene is very popular in DNA based identification of mosquito species. Timely reporting of mosquitoes is very crucial in vector born disease control. In this respect, in the present study, larvae of Culex quinquefasciatus species in Hyderabad, India was collected and reared to adults. Total DNA including mitochondrial DNA was isolated and partial mitochondrial COI gene was amplified. Interestingly, Hyderabad species sequence is found to be novel when compared to other geographical regions of the world, therefore, the same sequence was submitted to NCBI-Nucleotide database and accession number of JX08870 was assigned;to confirm it further, multiple alignment of Culex species was accomplished. Further, phylogenetic analysis was carried out to check the evolutionary pattern of Hyderabad species. In conclusion, partial mitochondrial COI gene of Hyderabad species is found to be novel and it can be used as DNA barcode for molecular characterization. Sequence alignment and phylogenetic analysis revealed that Hyderabad species is more close to UK species.

Hypersensitive Inhibition of the Proliferation of Cells with Mutated DNA Repair-Related Genes by the Catalytic Topoisomerase II Inhibitor 20-O-IngenolEZ

We previously reported that many ingenol compounds derived from Euphoria kansui exhibit topoisomerase inhibitory activity. 20-O-ingenolEZ in these compounds exerted inhibitory effects on both topoisomerase II (topo II) activity and cell proliferative activity. Topoisomerase II inhibitors can be divided into the poison and catalytic inhibitor types and 20-O-ingenolEZ is a catalytic inhibitor and inhibits topo IIα through inhibition of ATPase activity, but induces topo II-mediated DNA damage and apoptosis in BLM-/- DT40 cells through the induction of the DNA damage checkpoint, similar to the poison type inhibitor adriamycin. The ATPase inhibitor of topo II ICRF-193 also showed poison-like characteristics in the same cell line. However, the inhibitory effects of ICRF-193 on the proliferation of BLM-/- DT40 cells differed from those of 20-O-ingenolEZ, as did the specificity of its inhibition of the proliferation of other cell lines. 20-O-ingenolEZ showed hypersensitive inhibition of the proliferation of MCF-7 cells and BLM-/- DT40 cells with mutated DNA repair-related genes.

Effect of Chicken Akirin2 Gene on Immune Response Induced by VP2 DNA Vaccine of Infectious Bursal Dis-ease Virus

[ Abstracts ] In order to investigate the effect of chicken Akirin2 gene on the immune response induced by VP2 DNA vaccine of infectious bursal disease virus （IBDV）. [ Methods] The 14-day-old SPF chickens were immunized with recombinant plasmids expressing VP2 protein and Akirin2 protein, and strength- ened immunization was conducted at the 14＇~ day after the first immunization. Finally, test chickens were challenged with IBDVBC6-85 virulent strain. [ Resultss ] Test results showed that Akirin2 gene could enhance the specific immune response induced by VP2 DNA vaccine, improve the proliferation of peripheral blood lym- phocytes and ＇affect the expressing of cytokines TNF-a, IFN-Y, IL-1β, IL-2, IL-4, IL 6, IL-9, IL-10, IL-17 and IL-18. Effects of recombinant plasmids co-ex- pressing Akirin2 protein and VP2 protein on cytokine expression showed some differences with the recombinant plasmids expressing Akirir/2 protein or VP2 protein along. [ Conclusions] Chicken Akirin2 gene could significantly enhance the humoral immune response and cellular immune response induced by VP2 DNA vaccine of IBDV.

Non-invasive Prenatal Gene Diagnosis: Progress through Cell-free Fetal DNA and RNA in Maternal Plasma and Urine

Non-invasive prenatal gene diagnosis has been developed rapidly in the recent years, and numerous medical researchers are focusing on it. Such techniques could not only achieve prenatal diagnosis accurately, but also prevent tangential illness in fetuses and thus, reduce the incidence of diseases. Moreover, it is non-invasive prenatal gene diagnosis that prevents potential threaten and danger to both mothers and fetuses. Therefore, it is welcomed by clinical gynecologist and obstetrian, researchers of medical genetics, and especially, pregnancies. This review article touches briefly on the advanced development of using cell-free DNA, RNA in maternal plasma and urine for non-invasive prenatal gene diagnosis.

Aberrant DNA methylation in cervical carcinogenesis

Persistent infection wit h high-risk types of human papillomavirus(HPV) is known to cause cervical cancer;however,additional genetic and epigenetic alterations are required for progression from precancerous disease to invasive cancer.DNA methylation is an early and frequent molecular alteration in cervical carcinogenesis.In this review,we summarize DNA methylation within the HPV genome and human genome and identify its clinical implications.Methylation of the HPV long control region(LCR) and L1 gene is common during cervical carcinogenesis and increases with the severity of the cervical neoplasm.The L1 gene of HPV16 and HPV18 is consistently hypermethylated in invasive cervical cancers and can potentially be used as a clinical marker of cancer progression.Moreover,promoters of tumor suppressor genes(TSGs) involved in many cellular pathways are methylated in cervical precursors and invasive cancers.Some are associated with squamous cell carcinomas,and others are associated with adenocarcinomas.Identification of methylated TSGs in Pap smear could be an adjuvant test in cervical cancer screening for triage of women with high-risk HPV,atypical squamous cells of undetermined significance,or low grade squamous intraepithelial lesion(LSIL).However,consistent panels must be validated for this approach to be translated to the clinic.Furthermore,reversion of methylated TSGs using demethylating drugs may be an alternative anticancer treatment,but demethylating drugs without toxic carcinogenic and mutagenic properties must be identified and validated.