Mouse models of DNA double-strand break repair and neurological disease

The repair of DNA damage is essential for the prevention of disease. The DNA double-strand break (DSB) is a particularly hazardous lesion. DNA DSBs activate a coordinated cellular response involving cell cycle checkpoint activation and repair of the DNA break, or alternatively apoptosis. In the nervous system the inability to respond to DNA DSBs may lead to neurodegenerative disease or brain tumors. Therefore, understanding the DNA DSB response mechanism in the nervous system is of high importance for developing new treatments for neurodegeneration and cancer. In this regard, the use of mouse models represents an important approach for advancing our understanding of the biology of the DNA damage response in the nervous system.     

A distinct DNA methylation signature defines pediatric pre-B cell acute lymphoblastic leukemia

Pre-B cell acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and remains one of the highest causes of childhood mortality. Despite this, the mechanisms leading to disease remain poorly understood. We asked if recurrent aberrant DNA methylation plays a role in childhood ALL and have defined a genome-scale DNA methylation profile associated with the ETV6-RUNX1 subtype of pediatric ALL. Archival bone marrow smears from 19 children collected at diagnosis and remission were used to derive a disease specific DNA methylation profile. The gene signature was confirmed in an independent cohort of 86 patients. A further 163 patients were analyzed for DNA methylation of a three gene signature. We found that the DNA methylation signature at diagnosis was unique from remission. Fifteen loci were sufficient to discriminate leukemia from disease-free samples and purified CD34+ cells. DNA methylation of these loci was recurrent irrespective of cytogenetic subtype of pre-B cell ALL. We show that recurrent aberrant genomic methylation is a common feature of pre-B ALL, suggesting a shared pathway for disease development. By revealing new DNA methylation markers associated with disease, this study has identified putative targets for development of novel epigenetic-based therapies.     

Taxonomy of the Lyme disease spirochetes.

Morphology, physiology, and DNA nucleotide composition of Lyme disease spirochetes, Borrelia, Treponema, and Leptospira were compared. Morphologically, Lyme disease spirochetes resemble Borrelia. They lack cytoplasmic tubules present in Treponema, and have more than one periplasmic flagellum per cell end and lack the tight coiling which are characteristic of Leptospira. Lyme disease spirochetes are also similar to Borrelia in being microaerophilic, catalase-negative bacteria. They utilize carbohydrates such as glucose as their major carbon and energy sources and produce lactic acid. Long-chain fatty acids are not degraded but are incorporated unaltered into cellular lipids. The diamino amino acid present in the peptidoglycan is ornithine. The mole % guanine plus cytosine values for Lyme disease spirochete DNA were 27.3-30.5 percent. These values are similar to the 28.0-30.5 percent for the Borrelia but differed from the values of 35.3-53 percent for Treponema and Leptospira. DNA reannealing studies demonstrated that Lyme disease spirochetes represent a new species of Borrelia, exhibiting a 31-59 percent DNA homology with the three species of North American borreliae. In addition, these studies showed that the three Lyme disease spirochetes comprise a single species with DNA homologies ranging from 76-100 percent. The three North American borreliae also constitute a single species, displaying DNA homologies of 75-95 percent. Lyme disease spirochetes and Borrelia exhibited little or no DNA homology (0-2 percent) with the Treponema or Leptospira. Plasmids were present in the three Lyme disease spirochetes and the three North American borreliae.     

The Role of DNA Methylation in Lens Development and Cataract Formation

Epigenetics pertains to heritable alterations in genomic structural modifications without altering genomic DNA sequence. The studies of epigenetic mechanisms include DNA methylation, histone modifications, and microRNAs. DNA methylation may contribute to silencing gene expression which is a major mechanism of epigenetic gene regulation. DNA methylation regulatory mechanisms in lens development and pathogenesis of cataract represent exciting areas of research that have opened new avenues for association with aging and environment. This review addresses our current understanding of the major mechanisms and function of DNA methylation in lens development, age-related cataract, secondary cataract, and complicated cataract. By understanding the role of DNA methylation in the lens disease and development, it is expected to open up a new therapeutic approach to clinical treatment of cataract.     

Non-chemical method of DNA recovery and characterization of Mycobacterium avium subspecies paratuberculosis using IS 900 PCR

In the present study, two methods of DNA isolation-routine, traditional and standard DNA isolation protocol for Mycobacteria (Method 1) and a new non-chemicals and non-enzymes (physical) method (Method 2) of DNA recovery have been compared and evaluated in IS900 PCR for the specific detection of pathogen. Using the new Method 2, DNA has been recovered from few (1 - 3 colonies), extremely minute and stunted colonies. DNA, thus, isolated from these colonies (colonies PCR) and cultured for the first time from the cases of Crohn's disease in human beings, dairy cattle, raw milk and pasteurized commercial milk samples has been characterized in the present study. It is the first report from India.     

Mechanisms of non-canonical activation of ataxia telangiectasia mutated

ATM is a master regulator of the cellular response to DNA damage. The classical mechanism of ATM activation involves its monomerization in response to DNA double-strand breaks, resulting in ATM-dependent phosphorylation of more than a thousand substrates required for cell cycle progression, DNA repair, and apoptosis. Here, new experimental evidence for non-canonical mechanisms of ATM activation in response to stimuli distinct from DNA double-strand breaks is discussed. It includes cytoskeletal changes, chromatin modifications, RNA–DNA hybrids, and DNA single-strand breaks. Noncanonical ATM activation may be important for the pathology of the multisystemic disease Ataxia Telangiectasia.     

Archived Guthrie blood spots as a novel source for quantitative DNA methylation analysis

Sodium bisulfite treatment followed by PCR and DNA sequencing is widely considered the gold standard for the analysis of DNA methylation patterns. However, this technique generally requires substantial quantities of genomic DNA as starting material and is often associated with degradation of DNA. Here, we assess the feasibility of performing bisulfite sequencing on DNA isolated from 3-mm diameter punches of dried blood Guthrie spots. We demonstrate that it is possible to perform bisulfite sequencing from both freshly prepared and archived dried blood spots, using a combination of high purity DNA extraction and efficient bisulfite conversion. With the number of new technologies available for DNA methylation studies, we have extended this analysis and have successfully used a high-throughput mass spectrometry method for DNA methylation analysis on these samples. This provides a new source of material for epigenetic analysis of birth samples and provides an invaluable reference point to track temporal change in epigenetic profiles possibly linked with health and disease.     

DNA fingerprinting of Rattus norvegicus: a new approach in genetic analysis

Recent finding in highly effective DNA probes for RFLP testing (of hypervariable minisatellite DNA type) has led to the invention of DNA fingerprinting--the new technique of great value for identification of individuals, establishing biological kinship and studies in population genetics. We anticipate that DNA fingerprinting procedure with M13 phage DNA as a probe which we have developed earlier, makes it possible to apply new approach in genetic analysis--establishing, whether or not a particular locus is associated with the inheritance of genetic disease, by comparing the whole restriction fragment data from affected and unaffected animals. In this work, using the method described we characterized the Kroushynsky-Molodkina rat strain with hereditary disposition for epileptic attacks and performed comparative fingerprint analysis of these defective and normal rat genomes. The data obtained may hold some promises for further seeking the particular defective gene.     

Diagnosis of human heritable defects by recombinant DNA methods

Recombinant DNA methods provide highly sensitive means for the detection of DNA alterations that lead to human disease mutations. In this paper I shall illustrate the approaches currently available and discuss new technologies that show promise of replacing the present methods. Medical diagnosis by means of recombinant DNA methods has an expanding role in clinical medicine.     

Epigenetics in Alzheimer’s Disease: Perspective of DNA Methylation

Research over the years has shown that causes of Alzheimer’s disease are not well understood, but over the past years, the involvement of epigenetic mechanisms in the developing memory formation either under pathological or physiological conditions has become clear. The term epigenetics represents the heredity of changes in phenotype that are independent of altered DNA sequences. Different studies validated that cytosine methylation of genomic DNA decreases with age in different tissues of mammals, and therefore, the role of epigenetic factors in developing neurological disorders in aging has been under focus. In this review, we summarized and reviewed the involvement of different epigenetic mechanisms especially the DNA methylation in Alzheimer’s disease (AD), late-onset Alzheimer’s disease (LOAD), familial Alzheimer’s disease (FAD), and autosomal dominant Alzheimer’s disease (ADAD). Down to the minutest of details, we tried to discuss the methylation patterns like mitochondrial DNA methylation and ribosomal DNA (rDNA) methylation. Additionally, we mentioned some therapeutic approaches related to epigenetics, which could provide a potential cure for AD. Moreover, we reviewed some recent studies that validate DNA methylation as a potential biomarker and its role in AD. We hope that this review will provide new insights into the understanding of AD pathogenesis from the epigenetic perspective especially from the perspective of DNA methylation.     

精子DNA甲基化异常与男性不育

DNA甲基化／去甲基化是表观遗传学最重要的内容并可以控制基因的表达和印迹,越来越多的研究显示DNA甲基化异常与不育男性精子发生异常、特定肿瘤的发生、神经系统疾病、Rett综合征等有关。文章通过总结近来的相关研究资料来阐述精子发生过程中的DNA甲基化状态的改变,探讨精子DNA的甲基化异常与男性不育之间的联系,旨在为男性不育的治疗提供新的临床思路。     

血浆游离DNA检测在心血管系统疾病诊断中的应用

心血管疾病发病率和病死率居高不下,寻求敏感性高的生物学指标帮助早期诊断和改善预后意义重大.血浆游离DNA(cell-free DNA,cfDNA)是一类存在于血浆、尿液及其他体液中游离于细胞之外的双链DNA片段.血浆cfDNA水平在心血管疾病的早期明显升高,提示其可能是心血管疾病的潜在生物标志物.为了更深入理解cfDN...     

High-performance proteomics as a tool in biomarker discovery

Biomarkers allowing early detection of disease or therapy control have a huge influence in curing a disease. A wide variety of methods were applied to find new biomarkers. In contrast to methods focused on DNA or mRNA techniques, approaches considering proteins as potential biomarker candidates have the advantage that proteins are more diverse than DNA or RNA and are more reflective of a biological system. Here, we present an approach for the identification of new biomarkers relying on our experience from the past 10 years of proteomics, outlining a concept of "high-performance proteomics" This approach is based on quantitative proteome analysis using a sufficient number of clinical samples and statistical validation of proteomics data by independent methods, such as Western blot analysis or immunohistochemistry.     

The Human Specialized DNA Polymerases and Non-B DNA: Vital Relationships to Preserve Genome Integrity

In addition to the canonical right-handed double helix, DNA molecule can adopt several other non-B DNA structures. Readily formed in the genome at specific DNA repetitive sequences, these secondary conformations present a distinctive challenge for progression of DNA replication forks. Impeding normal DNA synthesis, cruciforms, hairpins, H DNA, Z DNA and G4 DNA considerably impact the genome stability and in some instances play a causal role in disease development. Along with previously discovered dedicated DNA helicases, the specialized DNA polymerases emerge as major actors performing DNA synthesis through these distorted impediments. In their new role, they are facilitating DNA synthesis on replication stalling sites formed by non-B DNA structures and thereby helping the completion of DNA replication, a process otherwise crucial for preserving genome integrity and concluding normal cell division. This review summarizes the evidence gathered describing the function of specialized DNA polymerases in replicating DNA through non-B DNA structures.     

Fellowship of the rings: the replication of kinetoplast DNA

Kinetoplastid protozoa such as trypanosomes and Leishmania are important because they cause human disease. These parasites are named after one of their most unusual features, a mitochondrial DNA known as kinetoplast DNA (kDNA). Unlike all other DNA in nature, kDNA comprises a giant network of interlocked DNA rings with a topology resembling that of medieval chain mail. The replication of the kDNA network is more complex than previously thought, and the discovery of new proteins involved in this process is currently the best approach for illuminating the replication mechanism.     

Recent patents of DNA methylation biomarkers in gastrointestinal oncology

Gastrointestinal malignancies are among the most common malignancies worldwide. Advances in technology and treatment have improved diagnosis and monitoring of these tumors. As a consequence, identification of new biomarkers that can be applied at different levels of disease is urgently needed. DNA methylation is a process in which cytosines acquire a methyl group in 5' position only if they are followed by a guanine. An emerging catalog of specific genes inactivated by DNA methylation in gastrointestinal tumors has been established. In this review we will give a brief overview of the main sources of DNA used to investigate methylation biomarkers and several related patents. One of these is related to multiple genes that predict the risk of development of esophageal adenocarcinoma. Another evaluated methylation status of 24 genes to find one frequently methylated in primary tumors as well as plasma samples from gastric cancer patients. Others patented the epigenetic silencing of miR-342 as a promissory biomarker for colorectal carcinoma. Thus the new field of DNA methylation biomarkers holds the promise of better methods for screening, early detection, disease progression and outcome predictor of therapy response in gastrointestinal oncology.     

Subregional assignment of the linked marker G8 (D4S10) for Huntington disease to chromosome 4p16.1-16.3.

The linked DNA marker for Huntington disease has recently been mapped to the short arm of chromosome 4 by somatic cell hybridization studies. Southern blot analysis of DNA from patients with Wolf-Hirschhorn syndrome (WHS) has suggested that the linked marker maps within the terminal 4p16 band. We have now accomplished subregional assignment of G8 (D4S10) to 4p16.1-16.3 using in situ hybridization techniques on two patients with nonoverlapping interstitial deletions of 4p. The mapping of G8 (D4S10) to a region deleted in patients with WHS will allow the application of new strategies for detecting DNA sequences closer to the locus for Huntington disease.     

Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation

Biofilms are a preferred mode of survival for many microorganisms including Vibrio cholerae, the causative agent of the severe secretory diarrhoeal disease cholera. The ability of the facultative human pathogen V. cholerae to form biofilms is a key factor for persistence in aquatic ecosystems and biofilms act as a source for new outbreaks. Thus, a better understanding of biofilm formation and transmission of V. cholerae is an important target to control the disease. So far the Vibrio exopolysaccharide was the only known constituent of the biofilm matrix. In this study we identify and characterize extracellular DNA as a component of the Vibrio biofilm matrix. Furthermore, we show that extracellular DNA is modulated and controlled by the two extracellular nucleases Dns and Xds. Our results indicate that extracellular DNA and the extracellular nucleases are involved in diverse processes including the development of a typical biofilm architecture, nutrient acquisition, detachment from biofilms and the colonization fitness of biofilm clumps after ingestion by the host. This study provides new insights into biofilm development and transmission of biofilm-derived V. cholerae.     

A method for fast database search for all k-nucleotide repeats.

A significant portion of DNA consists of repeating patterns of various sizes, from very small (one, two and three nucleotides) to very large (over 300 nucleotides). Although the functions of these repeating regions are not well understood, they appear important for understanding the expression, regulation and evolution of DNA. For example, increases in the number of trinucleotide repeats have been associated with human genetic disease, including Fragile-X mental retardation and Huntington's disease. Repeats are also useful as a tool in mapping and identifying DNA; the number of copies of a particular pattern at a site is often variable among individuals (polymorphic) and is therefore helpful in locating genes via linkage studies and also in providing DNA fingerprints of individuals. The number of repeating regions is unknown as is the distribution of pattern sizes. It would be useful to search for such regions in the DNA database in order that they may be studied more fully. The DNA database currently consists of approximately 150 million basepairs and is growing exponentially. Therefore, any program to look for repeats must be efficient and fast. In this paper, we present some new techniques that are useful in recognizing repeating patterns and describe a new program for rapidly detecting repeat regions in the DNA database where the basic unit of the repeat has size up to 32 nucleotides. It is our hope that the examples in this paper will illustrate the unrealized diversity of repeats in DNA and that the program we have developed will be a useful tool for locating new and interesting repeats.