Reduced non-CpG methylation is a potential epigenetic target after spinal cord injury

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury.To investigate the role of DNA methylation in spinal cord injury,we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points(day 0-42)after spinal cord injury in mice.Global DNA methylation levels,specifically non-CpG(CHG and CHH)methylation levels,decreased modestly following spinal cord injury.Stages post-spinal cord injury were classified as early(day 0-3),intermediate(day7-14),and late(day 28-42)based on similarity and hie rarchical cluste ring of global DNA methylation patterns.The non-CpG methylation level,which included CHG and CHH methylation levels,was markedly reduced despite accounting for a minor proportion of total methylation abundance.At multiple genomic sites,including the 5’untranslated regions,promoter,exon,intron,and 3’untranslated regions,the non-CpG methylation level was markedly decreased following spinal cord injury,whereas the CpG methylation level remained unchanged at these locations.Approximately one-half of the differentially methylated regions were located in intergenic areas;the other differentially methylated regions in both CpG and non-CpG regions were cluste red in intron regions,where the DNA methylation level was highest.The function of genes associated with differentially methylated regions in promoter regions was also investigated.From Gene Ontology analysis results,DNA methylation was implicated in a number of essential functional responses to spinal cord injury,including neuronal synaptic connection creation and axon regeneration.Notably,neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells.In summary,our work elucidated the dynamic pattern of DNA methylation in the spinal co rd following injury and identified reduced nonCpG methylation as an epigenetic target after spinal cord injury in mice.

Modified RNA with a Phosphate-Methylated Backbone. A Serious Omission in Our (Retracted) Study at HIV-1 RNA Loops and Integrated DNA. Specific Properties of the (Modified) RNA and DNA Dimers

After the recent publication in the Journal of Biophysical Chemistry entitled “Retracted HIV Study Provides New Information about the Status of the in Vitro Inhibition of DNA Replication by Back-bone Methylation”, it is of importance to review the results of Buck’s group on the synthesis and conformation analyses of phosphate-methylated RNAs in order to afford information on the absence of a further investigation with regard to this de facto acceptable approach. In fact these compounds belong to the very first group of RNAs with a modified neutral backbone by phosphatemethylation. In contrast to the corresponding phosphate-methylated DNAs with a frozen B-conformation, the phosphate-methylated RNAs show an A-conformation. The latter is a prerequisite for duplex formation with (complementary) (natural) RNA. A number of experiments support this fundamental statement. After the HIV study was retracted, the overall results concerning the phosphate-methylated RNAs were published without mentioning Buck’s initial proof of concept and his contributions. Generally, the (modified) dimer RNAs and DNAs possess a number of specific biophysical properties. A novel explanation is given for conflicting structural determinations.

恢复connexin26表达联合载酵母菌胞嘧啶脱氨酶自杀基因纳泡杀灭膀胱癌细胞

目的分析载双基因的阳离子纳泡联合超声靶向微泡破坏技术进行基因转染的有效性,探索恢复或上调膀胱癌细胞的缝隙连接蛋白connexin26(Cx26)表达,能否增强自杀基因系统(yeast cytosine deaminase/5-fluorocytosine,YCD/5-FC)的旁观者效应,提高杀灭肿瘤细胞的效率。方法阳离子纳泡结合超声辐照(US)转染人膀胱癌T24细胞,荧光显微镜及流式细胞仪观测转染效率;qRT-PCR和Western blot检测质粒转染后的mRNA或蛋白相对表达量。将实验分为无处理空白对照、载pc DNA3.1-EGFP纳泡组、载Cx26纳泡组、载YCD纳泡组、载YCD+Cx26纳泡组;通过流式细胞术观察恢复Cx26表达后对膀胱癌细胞凋亡的影响。结果 qRT-PCR、Western blot显示纳泡结合超声辐照成功将目的基因转染并有效表达。恢复Cx26表达后,载YCD+Cx26纳泡组的细胞凋亡率为(60.68±2.61)%,明显高于单载YCD纳泡组的(46.42±2.13)%,差异有统计学意义(P<0.01)。结论恢复缝隙连接蛋白Cx26表达,可改善细胞间通讯连接,加强自杀基因系统YCD/5-FC的旁观者效应,促进肿瘤细胞凋亡,提高杀灭膀胱癌细胞的效率。

Killing effects of cytosine deaminase gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro

OBJECTIVE: To evaluate the killing effects of the cytosine deaminase (CD) gene mediated by adenovirus vector on human pancreatic cancer cell lines in vitro. METHODS: The CD gene was cloned into pAdTrack-CMV-CD, and pAdTrack-CMV-CD and pAdEasy-l were recombinated in bacteria. The newly recombinated Ad-CD containing green fluoreseent protein (GFP) was propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic cancer cell lines Patu8988 and SW1990 were infected with this virus, then 5-FC was added. XTT assay was used to estimate relative numbers of viable cells. RESULTS: The positive clones were selected by using endonuclease to digest the combinatants and the concentration of viral liquids containing the CD gene was 2×1O^(11) pfu/ml. It was found that significant cytotoxic activities were possesscd by 5-FC for the CD gene transduced pancreatic cell lines, but little effects exerted on the nontransduced pancreatic carcinoma cells. CONCLUSIONS: The CD gene mediated by adenovirus with a high infectivity is efficient for gene therapy of pancreatic carcinoma cell lines. These data demonstrate the therapeutic efficacy of an enzyme prodrug strategy in experimental pancreatic cancer.

Preliminary Studies on Base Substitutions and Repair of DNA Mismatch Damage Stimulated by Low Energy N^+ Ion Beam Implantation in Escherichia coli

Ever since the low energy N+ ion beam has been accepted that the mutation effects of ionizing radiation are attributed mainly to direct or indirect damage to DNA. Evidences based on naked DNA irradiation in support of a mutation spectrum appears to be consistent, but direct proof of such results in vivo are limited. Using mutS, dam and/or dcm defective Eschericha coli imitator strains, an preliminary experimental system on induction of in vivo mutation spectra of low energy N+ ion beam has been established in this study. It was observed that the mutation rates of rifampicin resistance induced by N+ implantation were quite high, ranging from 9.2 x 10~8 to 4.9× 10~5 at the dosage of 5.2×1014 ions/cm2. Strains all had more than 90-fold higher mutation rate than its spontaneous mutation rate determined by this method. It reveals that base substitutions involve in induction of mutation of low energy nitrogen ion beam implantation. The mutation rates of mutator strains were nearly 500-fold (GM2929), 400-fold (GM5864) and 6-fold larger than that of AB1157. The GM2929 and GM5864 both lose the ability of repair DNA mismatch damage by virtue of both dam and dcm pathways defective (GM2929) or failing to assemble the repair complex (GM5864) respectively. It may explain the both strains had a similar higher mutation rate than GM124 did. It indicated that DNA cytosine methylase might play an important role in mismatch repair of DNA damage induced by N+ implantation. The further related research were also discussed.

IS HYPOMETHYLATION OF CELLULAR DNA A STEP REQUIRED IN THE INITIATION PROCESS OF CHEMICAL CARCINOGENESIS

The level of DNA methylation in carcinogen treat-ed cells were determined by HpaII restriction frag-ment length analysis of the newly replicated DNAslabelled with H-TdR 24 hr prior to harvesting aswell as by direct HPLC measurement of the 5-methyl-cytosine(~mC)contents of the cellular DNAs.

Highly efficient CRISPR-SaKKH tools for plant multiplex cytosine base editing

Base editing, as an expanded clustered regularly interspaced short palindromic repeats(CRISPR)-Cas genome editing strategy, permits precise and irreversible nucleotide conversion. SaKKH, an efficient variant of a Cas9 ortholog from Staphylococcus aureus(SaCas9), is important in genome editing because it can edit sites with HHHAAT protospacer adjacent motif(PAM) that the canonical Streptococcus pyogenes Cas9(SpCas9) or its variants(e.g. xCas9, Cas9-NG) cannot. However, several technical parameters of SaKKH involved base editors have not been well defined and this uncertainty limits their application. We developed an effective multiplex cytosine base editor(SaKKHn-pBE) and showed that it recognized NNARRT, NNCRRT, NNGRGT, and NNTRGT PAMs. Based on 27 targets tested, we defined technical parameters of SaKKHn-pBE including the editing window, the preferred sequence context, and the mutation type. The editing efficiency was further improved by modification of the SaKKH sgRNA. These advances can be applied in future research and molecular breeding in rice and other plants.

Research of total levels on DNA methylation in plant based on HPLC analysis

HPLC analysis is important for determination of total level on DNA methylation in plants. It can be used to help characterise epigenetic changes during growth, development and stress. HPLC methods have been optimised for mammalian and microbial DNA, but not for plants. This article examines several important factors in the HPLC analysis of plant DNA methylation including extraction and purification of DNA and HPLC conditions choice by using leaves of rice seedling. The experimental results showed that RNA of nucleic acid was removed by using RNase A. This study also identified critical components of HPLC analysis. With the optimized method of HPLC conditions, the better result was achieved in the chromatogram of cytosine and 5-methylcytosine in genomic DNA acid hydrolysis. The study would offer a comprehensive guide for the stringent analysis of DNA methylation in plants.

Analysis of DNA Cytosine Methylation on Cotton under Salt Stress

DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was reported that 6.5% of the whole cytosine residues in the nuclear DNA in

Theoretical investigations of collision dynamics of cytosine by low-energy (150-1000 eV) proton impact

Using a real-space real-time implementation of time-dependent density functional theory coupled to molecular dynamics(TDDFT-MD)nonadiabatically,we theoretically study both static properties and collision process of cytosine by 150-1000 eV proton impact in the microscopic way.The calculated ground state of cytosine accords well with experiments.It is found that proton is scattered in any case in the present study.The bond break of cytosine occurs when the energy loss of proton is larger than 22 eV and the main dissociation pathway of cytosine is the breaks of C1N2 and N8H10.In the range of 150 eV≤Ek≤360 eV,when the incident energy of proton increases,the excitation becomes more violent even though the interaction time is shortened.While in the range of 360 eV<Ek≤q 1000 eV,the excitation becomes less violent as the incident energy of proton increases,indicating that the interaction time dominates mainly.We also show two typical collision reaction channels by analyzing the molecular ionization,the electronic density evolution,the energy loss of proton,the vibration frequency and the scattering pattern detailedly.The result shows that the loss of electrons can decrease the bond lengths of C3N8 and C5N6 while increase the bond lengths of C4H11,C5H12 and C4C5 after the collision.Furthermore,it is found that the peak of the scattering angle shows a little redshift when compared to that of the loss of kinetic energy of proton.

Adenovirus-mediated tissue specific cytosine deaminase gene therapy for human hepatocellular carcinoma with different AFP expression level

Hepatocellular carcinoma (HCC) is one of the mostcommon cancers in the world, especially in East Asia.There is no standardized or effective strategy could beadapted routinely except of some early diagnosedpatients, and the prognosis is poor. In recent years, genetherapy has become a standard experimental approach fortreating cancers that have escaped conventionaltherapies. One such an approach is to confer the tumorcells with sensitivity to chemical reagents through

Therapeutic effects of combined suicide gene and granulocyte-macrophage colony stimulating factor gene transfer on erythroleukemia in mice

Adenoviruses harboring E. coli. cytosine deaminase(CD) gene (Ad-CD) and murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene(Ad-GM-CSF) were used for gene transfer in vivo.(C57BL/6 mice were inoculated subeutaneously with FBL-3 erythroleukemia cells and three days later treated withadenovirus injection at the site of tumor inoculation.

Combination of suicide gene and interleukin 2 gene transfer elicited potent antitumor effects on murine melanoma

Antitumor effects of combined transfer of suicidegene and cytokine gene were investigated in this report.Adenovirus harboring E.coli. cytosine deaminase (CD)gene (Ad-CD) and murine interleukin 2 (IL-2) gene(Ad-IL-2) were used for gene transfer in vitro and invivo. C57BL/6 mice were inoculated subcutaneously withB16F10 melanoma cells and three days later treated withadenovirus injection at the site of tumor inoculation.Significant inhibition of tumor growth was achieved

INHIBITION OF NF-κB ACTIVITY ENHANCED CYTOSINE ARABINOSIDE INDUCED APOPTOSIS IN LEUKEMIC CELL LINE HL60-N

Objective: To explore the effects of dexamethasone (DXM) and vincristine (VCR) on cytosine arabinoside (Ara-C) induced apoptosis and activation of nuclear factor-k-gene binding (NF-kB) in leukemic cell line HL60-n. Methods: Apoptosis of HL60-n cells was analysed by TdT-mediated X-dUTP nick and end labeling (TUNEL) and DNA electrophoresis. NF-kB activity of HL60-n cells was detected by electrophoretic mobility shift assay (EMSA). Results: There was slight activation of NF-kB in HL60-n cells without drug induction. Ara-C at 1 mmol/L significantly enhanced the activation of NF-kB in HL60-n cells. The level of NF-kB activation induced by DXM at 1 mmol/L or VCR at 0.1 mmol/L had no significant difference compared with that of the control group. However, in HL60-n cells pre-treated with 1 mmol/L of DXM or 0.1 mmol/L of VCR, the activation of NF-kB induced by 1 mmol/L of Ara-C was significantly suppressed with inhibition rates of 31.0% and 47.0%, respectively. The apoptosis rates of HL60-n cells induced by 1.0 mmol/L, 10 mmol/L and 100 mmot/L Ara-C were 45.003.16%, 61.883.40% and 77.624.75%, respectively. The apoptotic rates of HL60-n cells induced by DXM at 1 mmol/L or VCR at 0.1 mmol/L were similar to that of the control group. However, either DXM at 1 mmol/L or VCR at 0.l mmol/L could enhance the apoptosis of HL60-n cells induced by Ara-C at 1 mmol/L with rates of 39.1% and 59.2%, respectively. Conclusion: Ara-C can induce apoptosis and activation of NF-kB in HL60-n cells. The mechanism of increased apoptosis of HL60-n cells by DXM or VCR may be related to suppression of NF-kB activation.

Solution and Thermodynamic Studies on Binary and Ternary Complexes of Lanthanides

The interactions of La（ Ⅲ ）, Pr（ Ⅲ ）, Nd（ Ⅲ ）, Sm（ Ⅲ ）, Eu（Ⅲ）, Gd（ Ⅲ ）, Dy（ Ⅲ ）, and Yb（ Ⅲ ） with cytosine, 5-bromocytosine, 5-azacytosine and 5-flurocytosine as primary ligands （L） and guanine （A） as secondary ligand for both binary （ 1 ： 1 ） as well as ternary （ 1 ： 1 ： 1 ） systems were investigated by potentiometric equilibrium measurements at 25, 35 and 45 ℃ in aqueous solution （ionic strength, μ = 0.1 mol·dm^-3 NaNO3）. The stability constants of the binary （ 1 ： 1 ） and ternary （ 1 ： 1 ： 1 ） metal-ligand complexes follow the following order La 〈 Pr 〈 Nd 〈 Sm 〈 Eu 〉 Gd 〈 Dy 〉 Yb. Results also indicate that the ternary lanthanide complexes of La（Ⅲ）, Pr（Ⅲ）, Nd（Ⅲ）, Sm（Ⅲ）, Eu（Ⅲ）, Gd（Ⅲ）, Dy（Ⅲ）, and Yb（ Ⅲ ） are more stable than corresponding binary lanthanide complexes. The enthalpy （△Hf^o） and entropy （△Sf^o） changes for the formation of binary and ternary complexes were calculated from temperature coefficient data. The △△Sf^o values are positive for all the metal ligand systems. The negative △△Hf^o values indicate the extra stability of the ternary complexes by the exothermic enthalpy changes （△△Sf^o = △TSf^o - △BSf^o and △△Hf^o = △THf^o - △BHf^o where △TSf^o, △THf^o and △BSf^o, △BHf^o are the entropy and enthalpy values associated with the ternary and binary complexes, respectively）. It was also proposed that the guanine is bonded to metal ions through N1/C6 = O and N7 whereas cytosine and its derivatives are bonded through N3 atoms in ternary complexes.

Migration to gliomas of bone mesenchymal stem cells transfected with cytosine deaminase gene following transplantation in vivo

This experiment sought to observe the migration and distribution of bone mesenchymal stem cells transfected with the cytosine deaminase gone （BMSCs-CD/eGFP） after transplantation in vivo through three pathways. In addition, we examined the tropism of these cells to glioma. Intracranial C6 glioma models were established in Sprague-Dawley rats using an intracranial stereotactic inoculation method. When tumors were 7 days old, rats were inoculated with lx106 BMSCs-CD/eGFP cells via the tumor-bearing internal carotid artery, the contralateral hemisphere and the tumor-bearing glioma. Fluorescence microscopy revealed that BMSCs-CD/eGFP exhibited a strong capacity for migration to tumors. BMSCs-CD/eGFP transplanted via the tumor-bearing intemal carotid artery were observed to distribute in glioma tissues. BMSCs-CD/eGFP inoculated via the ipsilateral glioma mainly located within and at the edge of glioma tissues. BMSCs-CD/eGFP inoculated via the contralateral hemisphere mainly distributed at the proximal end of the tumor at the incubation site.

No consistent daily variation in DNA methylation detected in Populus nigra leaves by methylation-sensitive amplification polymorphism analysis

DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.

PtRu nanoparticles supported on noble carbons for ethanol electrooxidation

In this work,three cytosine derived nitrogen doped carbonaceous materials(noble carbons,NCs)with different atomic C/N ratios and porous networks have been synthesized and used as supports for Pt Ru electrocatalysts in the ethanol oxidation reaction(EOR)for clean hydrogen production.Both,the metal phase and the carbon support play critical roles in the electrocatalysts final performance.Lower NPs size distribution was obtained over supports with low atomic C/N ratios(i.e.,4 and 6)and defined porosity(i.e.,1701 m^(2)g^(-1)for Pt Ru/CNZ and 1834 m^(2)g^(-1)for Pt Ru/CLZ,respectively).In contrast,a lower C/N ratio and poor porous network(i.e.,65 m^(2)g^(-1),Pt Ru/CLK)led to the largest particle size and fostered an increase of the alloying degree between Pt and Ru NPs(i.e.,3%for C/N~6 and 28%for C/N~3).Electrochemical active surface area was found to increase with decreasing NPs size and the alloy extent,due to a higher availability of Pt active sites.Accelerated degradation tests showed that Pt Ru/NCs outperform similar to Pt Ru NPs on commercial carbon pointing at the stabilizing effect of NCs.Pt Ru/CNZ exhibited the best electrochemical performance(i.e.,69.1 m A mgPt-1),outperforming Pt Ru/CLZ and Pt Ru/CLK by3-and 9-fold,respectively,due to a suitable compromise between particle sizes,degree of alloy,textural properties and elemental composition.Best anodes were scaled-up to a proton exchange membrane cell and Pt Ru/CNZ was proved to provide the best electrocatalytic activity(262 m A cm^(-2)and low energy requirements),matching the values obtained by the state of the art of EOR electrocatalysts.

Theoretical Studies on the Interaction between Metal Cations and Cytosine, Guanine

The interaction of tetra- and hexa-coordinated compounds of cytosine(C) and guanine(G) with metal cations Ca(2+), Mg(2+), Mn(2+), Ni(2+), Cu(2+), and Zn(2+) have been calculated by using the B3LYP/6-31G method at the 6-31G(d, p) basis set, while the remaining coordination bonds are saturated by water molecules ((H(2)O)(4)). All geometries were optimized without symmetry restrictions. Comparing the interaction energies we obtained the orders of selectivity of C and G for the above metal ions as follows: (a)Cu(2+)>(a)Ni(2+)>(a)Mg(2+)>(a)Mg(2+)>(b)Cu(2+)>(a)Mn(2+)>(b)Zn(2+)> Ni(2+) and (a)Cu(2+)>(a)Ni(2+> a)Zn(2+)>(a)Mg(2+)>(b)Cu(2+)>(a)Mn(2+)>(b)Zn(2+)>(b)Ni(2+) respectively ((a,b) represent tetra- and hexa-coordinated, respectively), which are in good agreement with the experimental facts. Interaction energies of complexes provide a comparatively reliable quantification of the selectivity of dimethyl phosphate anion for the studied metal ions. In addition, the influence of coordination number and coordination structure on the interaction energy and the variation of ionic energy were discussed sufficiently. After analyzing the interaction energies of two kinds of complexes, the 'mutual selectivity'as well as the nature of the interaction between metal ions and ligands was revealed.

Inhibitory effects of cytosine deaminase gene-transfected bone marrow mesenchymal stem cells on glioma cell proliferation

Bone marrow mesenchymal stem cells were isolated from C57BL mice, transfected with the cytosine deaminase （CD） gene using a lentivirus vector and co-cultured with C6 glioma cells to verify anti-tumor effects of bone marrow mesenchymal stem cells carrying CD genes. C57MSC-CD/eGFP cells converted 5-fluorocytosine to 5-fluorouracil and exhibited significant inhibition of proliferation and apoptosis in C6 glioma cells. C57MSC-CD/eGFP cells were then implanted into rat models of brain C6 glioma. Rats were also intraperitoneally injected with 5-fluorocytosine after 7 days. MSC-CD/eGFP cells were irregularly distributed at the margin of the glioma, as well as encased and reduced the volume of the glioma. CD-transfected bone marrow mesenchymal stem cells inhibit the in vivo growth and in vitro proliferation of glioma.