Mapping the epigenetic modifications of DNA and RNA

Over 17 and 160 types of chemical modifications have been identified in DNA and RNA, respectively. The interest in understanding the various biological functions of DNA and RNA modifications has lead to the cutting-edged fields of epigenomics and epitranscriptomics. Developing chemical and biological tools to detect specific modifications in the genome or transcriptome has greatly facilitated their study. Here, we review the recent technological advances in this rapidly evolving field. We focus on high-throughput detection methods and biological findings for these modifications, and discuss questions to be addressed as well. We also summarize third-generation sequencing methods, which enable long-read and single-molecule sequencing of DNA and RNA modification.

Uptake of DNA by cancer cells without a transfection reagent

BACKGROUND: Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. METHODS: A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer) and THLE3 (normal liver cells) after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH)" from Mirus (USA). RESULTS: The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. CONCLUSIONS: In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA fragments directly from the environment without the aid of the transfecting reagent.

A Family With a Hemoglobin E Variant Including a Thai Immigrant Woman in Korea

No abstract available.

A Case of Chronic Myeloid Leukemia With Rare Variant ETV6/ABL1 Rearrangement

No abstract available.

Latest progress in postmortem interval estimation / 法医学杂志

Accurate estimation of the postmortem interval (PMI) has been one of the most important and complicated issues in the forensic practice. In order to provide novel perspectives for the future research concerning PMI, the advantages and disadvantages of related traditional methods, postmortem degradation of nucleic acid and tissue, the componential change of vitreous humor and histological biochemistry since 2002 have been introduced and compared in this review.

DNA Double Strand Breaks in Gastric Epithelium with Helicobacter pylori Infection / 대한소화기학회지

BACKGROUND/AIMS: DNA double strand breaks (DSB) is one of the critical types of DNA damage. If unrepaired, DSB is accumulated in the nucleus of cells, the cells become apoptotic or transform to tumor by way of genomic instability. Some of malignant cancers and its premalignant lesions were proven to have DSB in their nuclei. There was no report that Helicobacter pylori (H. pylori), the gastric carcinogen, induce DNA DSB in gastric epithelium in vivo. The aim of this study was to investigate whether H. pylori induce DSB in the gastric epithelial cells of chronic gastritis. METHODS: Immunohistochemical stains were performed for the DSB markers, phospho-53BP1 and gammaH2AX, in the gastric epithelium derived from 44 peptic ulcer disease patients before and after H. pylori eradication. DNA fragmentation assay was performed in the cell line to investigate the DNA damage by H. pylori infection. RESULTS: The mean expression score of gammaH2AX was significantly higher in the H. pylori infected gastric epithelium as compared to the H. pylori eradicated gastric epithelium (8.8+/-5.5 vs. 6.2+/-5.3 respectively; p=0.008). The expression score of phospho-53BP1 between before and after eradication of H. pylori was not statistically different, but tended to be higher in H. pylori infection. DNA fragmentation was developed significantly more in the cell lines after infection with H. pylori. CONCLUSIONS: DSB of DNA damage was typical feature of H. pylori infection in the gastric epithelium.

Application of nucleic acids and proteins in estimation of postmortem interval / 法医学杂志

Accurate estimation of postmortem interval (PMI) is one of the most important and difficult issues in forensic medicine. After death, the tissues autolyze and biomacromolecules degrade. DNA concentration decreases gradually with linear relationship with PMI. The housekeeping gene mRNA, for example beta-actin, GAPDH, has certain stability and can be used to PMI estimation as internal standard. This paper reviews the research progress and problems about DNA, RNA and proteins in the estimation of PMI in order to provide guidance for forensic pathology.

Decay in intact DNA recovery in blood samples kept at room temperature.

Blood Samples were kept at room temperature for a period of 3-6 months at room temperature to know the amount of quantitative DeoxyriboNucleic Acid [DNA] recovery from these samples. We are able to recover good amount of DNA for about first 3-6 weeks after which the DNA is decreased drastically and after two months there hardly any chance of intact DNA recovery from these samples. It has been concluded that blood samples recovered from scene of crime after about 1-2 months is a waste. The samples must be recovered as early as possible to recover intact DNA from them. The samples must be collected within 1-2 months from scene of crime until and unless the climate is cold enough to increases decay time. This study is very useful for the investigating authorities which can make errors while collecting blood samples for DNA analysis.

Construction and application of fluorescence labeled multiplex typing system for 3 new miniSTR loci / 法医学杂志

OBJECTIVE@#To establish a miniSTR multiplex set including three STR loci unlinked from the CODIS loci: D1S1676, D6S1274 and D17S1299, to generate amplified fragment less than 115 bp in size and to study the genotype of degraded DNA samples.@*METHODS@#After amplification with different fluorescence labeled primers, the amplified products from 100 unrelated individual and 2 highly degraded specimens were analyzed by 310 Genetic Analyzer.@*RESULTS@#Three miniSTR loci were determined by fluorescence-labeled multiplex-PCR technique. Each locus was successfully genotyped in all 100 samples. In D1S1676, D6S1274 and D17S1299 loci, 9, 9, 7 alleles and 27, 23, 18 genotypes were observed respectively. The distribution of genotype for three miniSTR loci in Chengdu Han population was in accordance with Hardy-Weinberg equilibrium. The combined exclusion probability and the combined discrimination power of the three STR loci in Chengdu Han population were 0.9991 and 0.9160 respectively.@*CONCLUSION@#This miniSTR multiplex set could be used in individual identification and paternity test. It also provides a new method in the analysis of degraded DNA sample.

DNA degradation of porcine retinal cells for estimation of postmortem interval / 法医学杂志

OBJECTIVE@#To investigate DNA degradation of porcine retinal cells by single cell gel electrophoresis (SCGE) for estimation of postmortem interval (PMI).@*METHODS@#Degradation of retinal cells was observed by SCGE at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24h after death respectively, under the environmental conditions of being kept in dark place as well as a controlled temperature of (15 +/- 2) degrees C and humidity of (50 +/- 5)%. The comet pictures were captured by fluorescence microscope and analyzed by single cell gel electrophoresis (IMI 1.0).@*RESULTS@#From 2h to 24h postmortem, the degree of degradation of retinal DNA increased with the prolongation of PMI. The postmortem regression functions of head DNA%, L(T)/L(H), I(T)/I(H) were y = 92.227-5.188 x + 0.019 x2 + 0.001 x3 (R2 = 0.971), y = 0.035e(0.191x) (R2 = 0.947), y = 0.099e(0.264x) (R2 = 0.955), respectively.@*CONCLUSION@#The examination of retinal cell DNA degradation by SCGE is useful for estimating PMI.

DNA degradation in nucleolus of skeletal muscle, heart, liver, kidney and brain in mice after death / 法医学杂志

OBJECTIVE@#To study the change of DNA degradation in nucleolus of mice organs and its relationship with the postmortem interval, and to investigate a new accurate method to estimate the postmortem interval.@*METHODS@#Eight parameters of cell nuclei were chosen, including the head DNA level, the tail DNA level, the head radius, the tail length, the tail moment, the Olive moment, the head area and the tail area. The changes of DNA degradation were analyzed in skeletal muscle, myocardium, liver, kidney and brain in mice at different intervals (0-72 h postmortem) by using single-cell gel electrophoresis and fluorescent microscope connected with auto-analysis-image system.@*RESULTS@#The tail DNA level, the tail length, the tail moment, the Olive moment and the tail area showed an increasing tendency. The head DNA level, the head radius and the head area showed a decreasing tendency within 72h postmortem in mice. A quadratic regression equation (P < 0.001) and multiple regression equation of DNA degradation tendency were established (P < 0.000 1).@*CONCLUSION@#The regression equations established can be used as a new method for estimating postmortem interval in forensic practice.

Disruption of Microtubules Sensitizes the DNA Damage-induced Apoptosis Through Inhibiting Nuclear Factor kappaB (NF-kappaB) DNA-binding Activity

The massive reorganization of microtubule network involves in transcriptional regulation of several genes by controlling transcriptional factor, nuclear factor-kappa B (NF-kappaB) activity. The exact molecular mechanism by which microtubule rearrangement leads to NF-kappaB activation largely remains to be identified. However microtubule disrupting agents may possibly act in synergy or antagonism against apoptotic cell death in response to conventional chemotherapy targeting DNA damage such as adriamycin or comptothecin in cancer cells. Interestingly pretreatment of microtubule disrupting agents (colchicine, vinblastine and nocodazole) was observed to lead to paradoxical suppression of DNA damage-induced NF-kappaB binding activity, even though these could enhance NF-kappaB signaling in the absence of other stimuli. Moreover this suppressed NF-kappaB binding activity subsequently resulted in synergic apoptotic response, as evident by the combination with Adr and low doses of microtubule disrupting agents was able to potentiate the cytotoxic action through caspase-dependent pathway. Taken together, these results suggested that inhibition of microtubule network chemosensitizes the cancer cells to die by apoptosis through suppressing NF-kappaB DNA binding activity. Therefore, our study provided a possible anti-cancer mechanism of microtubule disrupting agent to overcome resistance against to chemotherapy such as DNA damaging agent.

Kinetics of oxidation of adenosine by tert-butoxyl radicals: Protection and repair by chlorogenic acid.

The rates of oxidation of adenosine and chlorogenic acid by tert-butoxyl radicals (t-BuO-) were studied by measuring the absorbance of adenosine at 260 nm and chlorogenic acid at 328 nm spectrophotometrically. t-BuO- radicals were generated by the photolysis of tert-butyl hydroperoxide (t-BuOOH) in presence of tert-butyl alcohol to scavenge OH. radicals. The rates and the quantum yields() of oxidation of chlorogenic acid by t-BuO-radicals were determined in the absence and presence of varying concentrations of adenosine. An increase in the concentration of adenosine was found to decrease the rate of oxidation of chlorogenic acid, suggesting that adenosine and chlorogenic acid competed for t-BuO. radicals. From competition kinetics, the rate constant of chlorogenic acid reaction with t-BuO- was calculated to be 3.20 109 dm3 mol-1 s-1. The quantum yields (expt) were calculated from the experimentally determined rates of oxidation of chlorogenic acid under different experimental conditions. Assuming that chlorogenic acid acts as a scavenger of t-BuO- radicals only, the quantum yields (cal) were theoretically calculated. expt and cal values suggested that chlorogenic acid not only protected adenosine from t-BuO- radicals, but also repaired adenosine radicals, formed by the reaction of adenosine with t-BuO- radicals.

Free radical induced oxidative damage to DNA: relation to brain aging and neurological disorders.

Free radicals are produced in cells by cellular metabolism and by exogenous agents. These species react with biomolecules in cells and one of the important targets is DNA. This kind of damage, often referred to as oxidative DNA damage, has consequences in various organs and particularly in brain, in view of its high metabolic activity and oxygen consumption. The consequences include mutagenesis of various kinds ranging from simple oxidation of bases to large deletions through single and double strand breaks. In brain, because of its post-mitotic nature, oxidative damage to DNA is seen more often at the level of bases. A major route for repairing oxidative damage to bases is base excision repair (BER). It is increasingly becoming apparent that defects in repairing oxidative DNA damage can lead to a number of neurological disorders like Alzheimer and Parkinson. Our recent studies have clearly demonstrated that BER is highly compromised in brain cells with increasing age and this could well be one of the major causative factors for normal aging and the associated deteriorating mental conditions, including certain neurological abnormalities.

Aldosterone-induced TGF-beta1 Expression is Regulated by Mitogen-Activated Protein Kinases and Activator Protein-1 in Mesangial Cells

Aldosterone has been shown to stimulate renal TGF-beta1 expression. However, the mechanisms for aldosterone-induced TGF-beta1 expression have not been clearly determined in mesangial cells. We examined the role of extracellular-signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and activator protein- 1 (AP-1) in the aldosterone-induced TGF-beta1 expression in rat mesangial cells. TGF-beta1 protein in the conditioned medium released from rat mesangial cells was measured by sandwich ELISA, TGF-beta1 mRNA expression was analyzed by Northern blotting, AP-1 DNA binding activity was measured by EMSA and the ERK1/2, JNK activity was analyzed by western blotting. Aldosterone significantly stimulated TGF-beta1 protein production and TGF-beta1 mRNA expression in mesangial cells in a dose-dependent manner. Aldosterone significantly increased AP-1 DNA binding activity in mesangial cells. Pre-treatment of cells with AP-1 inhibitor, curcumin, blocked aldosterone-induced AP-1 DNA binding activity as well as aldosterone-induced TGF-beta1 production. Aldosterone increased phosphorylation of ERK1/2 and JNK in mesangial cells. Pre-treatment of cells with ERK1/2 inhibitor, PD98059, or JNK inhibitor, SP600125 significantly inhibited aldosterone-induced ERK1/2 and JNK activity and subsequently TGF-beta1 production, respectively. We conclude that aldosteroneinduced TGF-beta1 expression in mesangial cells is regulated by the ERK1/ 2, JNK and AP-1 intracellular signaling pathways.

A tribute to Fritz-Albert Popp on the occasion of his 70th birthday.

On May 11, 2008 the German biophysicist Professor Fritz-Albert Popp will celebrate his 70th birthday. This is a welcome occasion to pay tribute to the scientific achievements and human qualities of a scientist whose merits as one of the founders of biophotonics and as a pioneer of quantum biophysics increasingly find appreciation internationally.

Characterization of Streptococcus pneumoniae ophthalmic, systemic & commensal isolates by pulsed-field gel electrophoresis & ribotyping.

BACKGROUND & OBJECTIVE: Streptococcus pneumoniae is common in ocular and systemic infections and is a part of normal nasopharyngeal flora. Very few studies regarding genetic analysis of S. pneumoniae isolates causing eye infections are available. This study was undertaken to do pulse field gel electrophoresis (PFGE) analysis and ribotyping of S. pneumoniae isolates obtained from eye infections, systemic infections and nasopharyngeal flora. METHODS: Sixty one well characterized S. pneumoniae isolates (38 from ophthalmic infections, 9 from systemic infections and 14 commensals) were characterized using PFGE of the whole genome after SmaI, restriction enzyme digestion and conventional ribotyping using Escherichia coli rRNA operon as the probe. Phylogenetic tree was drawn using unweighted pair group method analysis (UPGMA). RESULTS: The 38 S. pneumoniae isolates from eye infections belonging to 15 serotypes were placed in to 11 PFGE types and 15 ribotypes. The 9 systemic isolates (7 seotypes) were distributed in 7 PFGE types and 6 ribotypes. The 14 commensal isolates were placed in 11 serotypes, 5 PFGE types and 6 ribotypes. Most of the PFGE types and ribotypes consisting of ocular isolates also contained systemic and commensal isolates. INTERPRETATION & CONCLUSION: Considerable genetic similarity was observed between the isolates from ocular and systemic infections and those colonized in nasopharynx. PFGE analysis could differentiate majority of the isolates according to site of infections. There was a considerable DNA polymorphism within the studied bacterial population.

The correlation between the DNA content in human thyroid follicular epithelial cells and the postmortem interval / 法医学杂志

OBJECTIVE@#To study the relationship between the DNA content in follicular epithelial cells of the human thyroid and postmortem interval (PMI).@*METHODS@#Changes of the DNA content in thyroid follicular epithelial cells at different PMI were determined by Methyl Green-Pyronin (MGP) stain combined with an image analysis technique.@*RESULTS@#The average DNA content in the thyroid follicular epithelial continued to decrease with increased PMI. The coefficient of determination for DNA content with average gray, aimed area, aimed area ratio and positive index in follicular epithelial cells were 0.960, 0.987, 0.988 and 0.990, respectively.@*CONCLUSION@#There is an apparent correlation between the average DNA content of follicular epithelial cells and the PMI. MGP stain combined with an image analysis technique seems to be a useful means to study the degradation of DNA in thyroid follicular epithelial cells.

Recent advancement in relationship between DNA degradation and postmortem interval / 法医学杂志

Determination of postmortem interval (PMI) is one of the most valuable subjects in forensic practice. It, however, is often very difficult to accurately determine the PMI in daily practice. Forensic DNA technology has recently been used to estimate the PMI. It has certain advantage to traditional methods. This article reviews this technology with respect to its invention, development, advantage, disadvantage, and potential future applications with emphasis on correlation of DNA degradation and PMI.

Relationship between the postmortem interval and nuclear DNA changes of heart muscular cells in mice / 法医学杂志

OBJECTIVE@#To study the changes of DNA degradation in cells of rats and its relationship with the postmortem interval.@*METHOD@#8 parameters of cell nuclear (including the tail length, the head radius, the percentage of head DNA, the percentage of tail DNA, the tail moment, the olive moment, the head area and the tail area) were chosen to study their changes in the procedure of DNA degradation in myocardium cells in 111 rats at different postmortem interval from 0 to 72 h by using single-cell gel electrophoresis (SCGE) technology and fluorescent microscope combined with auto-analysis-image system method.@*RESULTS@#An evident comet tailing was observed in DNA of myocardium cells after electrophoresis, and their changes in all these 8 parameters of cell nuclear were greatly associated with the extension of postmortem interval, which indicate the degradation rate and degree of DNA in the nuclear has a close relationship with postmortem interval in the periods from 0 to 72 h in rats and significant difference were found with those groups (P < 0.001).@*CONCLUSION@#The equations, which were concluded from our study, indicate the close relationship of degradation rate and degree of DNA in the nuclear with postmortem interval from 0 to 72 h, and provide an objective and exact new way to estimate the postmortem interval.