Journal of Nucleic Acids The latest articles from Hindawi © 2017 , Hindawi Limited . All rights reserved. The 2D Structure of the T. brucei Preedited RPS12 mRNA Is Not Affected by Macromolecular Crowding Sun, 18 Jun 2017 00:00:00 +0000 Mitochondrial transcript maturation in African trypanosomes requires RNA editing to convert sequence-deficient pre-mRNAs into translatable mRNAs. The different pre-mRNAs have been shown to adopt highly stable 2D folds; however, it is not known whether these structures resemble the in vivo folds given the extreme “crowding” conditions within the mitochondrion. Here, we analyze the effects of macromolecular crowding on the structure of the mitochondrial RPS12 pre-mRNA. We use high molecular mass polyethylene glycol as a macromolecular cosolute and monitor the structure of the RNA globally and with nucleotide resolution. We demonstrate that crowding has no impact on the 2D fold and we conclude that the MFE structure in dilute solvent conditions represents a good proxy for the folding of the pre-mRNA in its mitochondrial solvent context. W.-Matthias Leeder, Stephan Voskuhl, and H. Ulrich Göringer Copyright © 2017 W.-Matthias Leeder et al. All rights reserved. Selection of DNA Aptamers for Ovarian Cancer Biomarker CA125 Using One-Pot SELEX and High-Throughput Sequencing Thu, 09 Feb 2017 00:00:00 +0000 CA125 is a mucin glycoprotein whose concentration in serum correlates with a woman’s risk of developing ovarian cancer and also indicates response to therapy in diagnosed patients. Accurate detection of this large, complex protein in patient samples is of great clinical relevance. We suggest that powerful new diagnostic tools may be enabled by the development of nucleic acid aptamers with affinity for CA125. Here, we report on our use of One-Pot SELEX to isolate single-stranded DNA aptamers with affinity for CA125, followed by high-throughput sequencing of the selected oligonucleotides. This data-rich approach, combined with bioinformatics tools, enabled the entire selection process to be characterized. Using fluorescence anisotropy and affinity probe capillary electrophoresis, the binding affinities of four aptamer candidates were evaluated. Two aptamers, CA125_1 and CA125_12, both without primers, were found to bind to clinically relevant concentrations of the protein target. Binding was differently influenced by the presence of Mg2+ ions, being required for binding of CA125_1 and abrogating binding of CA125_12. In conclusion, One-Pot SELEX was found to be a promising selection method that yielded DNA aptamers to a clinically important protein target. Delia J. Scoville, Tae Kyu Brian Uhm, Jamie A. Shallcross, and Rebecca J. Whelan Copyright © 2017 Delia J. Scoville et al. All rights reserved. Systemic Identification of Hevea brasiliensis EST-SSR Markers and Primer Screening Mon, 23 Jan 2017 00:00:00 +0000 This research aimed to systematically identify and preliminarily validate the Hevea brasiliensis expressed sequence tag (EST) information using Simple Sequence Repeat (SSR) and provide evidence for further development of SSR molecular marker. The definition of general SSR features of Hevea EST splicing sequences and development of SSR primers founded the basis of diversity analysis and variety identification for Hevea tree resource. 1134 SSR loci were identified in the EST splicing sequence and distributed in 840 Unigene. The occurrence rate of SSR loci was 23.9%, and the average distribution distance of EST-SSR was 2.59 kb. The major repeat type was mononucleotide repeat motif, which accounted for 38.89%, while the corresponding value was 36.95% for dinucleotide repeat motif and 18.17% for trinucleotide repeat motif; the proportion of other motifs was only 5.99%. The superior repeat motifs for mononucleotide, dinucleotide, and trinucleotide were A/T, AG/CT, and AAG/CTT, respectively. 739 pair of primers were designed for 1134 SSR loci. PCR amplification was performed on Hevea Reyan5-11, Reyan87-6-47, and PR107, and 180 pairs of primers were selected which were able to amplify polymorphism bands. Benjun Hou, Suping Feng, and Yaoting Wu Copyright © 2017 Benjun Hou et al. All rights reserved. Cell-SELEX Identifies a “Sticky” RNA Aptamer Sequence Tue, 17 Jan 2017 09:24:22 +0000 Cell-SELEX is performed to select for cell binding aptamers. We employed an additional selection pressure by using RNAse to remove surface-binding aptamers and select for cell-internalizing aptamers. A common RNA sequence was identified from independent cell-SELEX procedures against two different pancreatic cancer cell lines, indicating a strong selection pressure towards this sequence from the large pool of other available sequences present in the aptamer library. The aptamer is not specific for the pancreatic cancer cell lines, and a similar sequence motif is present in previously published internalizing aptamers. The identified sequence forms a structural motif that binds to a surface protein, which either is highly abundant or has strong affinity for the selected aptamer sequence. Deselecting (removing) this sequence during cell-SELEX may increase the probability of identifying aptamers against cell type-specific targets on the cell surface. Partha Ray and Rebekah R. White Copyright © 2017 Partha Ray and Rebekah R. White. All rights reserved. Overexpression of PCNA Attenuates Oxidative Stress-Caused Delay of Gap-Filling during Repair of UV-Induced DNA Damage Sun, 01 Jan 2017 13:22:48 +0000 UVC irradiation-caused DNA lesions are repaired in mammalian cells solely by nucleotide excision repair (NER), which consists of sequential events including initial damage recognition, dual incision of damage site, gap-filling, and ligation. We have previously shown that gap-filling during the repair of UV-induced DNA lesions may be delayed by a subsequent treatment of oxidants or prooxidants such as hydrogen peroxide, flavonoids, and colcemid. We considered the delay as a result of competition for limiting protein/enzyme factor(s) during repair synthesis between NER and base excision repair (BER) induced by the oxidative chemicals. In this report, using colcemid as oxidative stress inducer, we showed that colcemid-caused delay of gap-filling during the repair of UV-induced DNA lesions was attenuated by overexpression of PCNA but not ligase-I. PCNA knockdown, as expected, delayed the gap-filling of NER but also impaired the repair of oxidative DNA damage. Fen-1 knockdown, however, did not affect the repair of oxidative DNA damage, suggesting repair of oxidative DNA damage is not of long patch BER. Furthermore, overexpression of XRCC1 delayed the gap-filling, and presumably increase of XRCC1 pulls PCNA away from gap-filling of NER for BER, consistent with our hypothesis that delay of gap-filling of NER attributes the competition between NER and BER. Yi-Chih Tsai, Yi-Hsiang Wang, and Yin-Chang Liu Copyright © 2017 Yi-Chih Tsai et al. All rights reserved. Role of Eukaryotic Initiation Factors during Cellular Stress and Cancer Progression Mon, 19 Dec 2016 11:19:29 +0000 Protein synthesis can be segmented into distinct phases comprising mRNA translation initiation, elongation, and termination. Translation initiation is a highly regulated and rate-limiting step of protein synthesis that requires more than 12 eukaryotic initiation factors (eIFs). Extensive evidence shows that the transcriptome and corresponding proteome do not invariably correlate with each other in a variety of contexts. In particular, translation of mRNAs specific to angiogenesis, tumor development, and apoptosis is altered during physiological and pathophysiological stress conditions. In cancer cells, the expression and functions of eIFs are hampered, resulting in the inhibition of global translation and enhancement of translation of subsets of mRNAs by alternative mechanisms. A precise understanding of mechanisms involving eukaryotic initiation factors leading to differential protein expression can help us to design better strategies to diagnose and treat cancer. The high spatial and temporal resolution of translation control can have an immediate effect on the microenvironment of the cell in comparison with changes in transcription. The dysregulation of mRNA translation mechanisms is increasingly being exploited as a target to treat cancer. In this review, we will focus on this context by describing both canonical and noncanonical roles of eIFs, which alter mRNA translation. Divya Khandige Sharma, Kamiko Bressler, Harshil Patel, Nirujah Balasingam, and Nehal Thakor Copyright © 2016 Divya Khandige Sharma et al. All rights reserved. Copy Number Variation of UGT 2B Genes in Indian Families Using Whole Genome Scans Tue, 22 Mar 2016 12:40:56 +0000 Background and Objectives. Uridine diphospho-glucuronosyltransferase 2B (UGT2B) is a family of genes involved in metabolizing steroid hormones and several other xenobiotics. These UGT2B genes are highly polymorphic in nature and have distinct polymorphisms associated with specific regions around the globe. Copy number variations (CNVs) status of UGT2B17 in Indian population is not known and their disease associations have been inconclusive. It was therefore of interest to investigate the CNV profile of UGT2B genes. Methods. We investigated the presence of CNVs in UGT2B genes in 31 members from eight Indian families using Affymetrix Genome-Wide Human SNP Array 6.0 chip. Results. Our data revealed >50% of the study members carried CNVs in UGT2B genes, of which 76% showed deletion polymorphism. CNVs were observed more in UGT2B17 (76.4%) than in UGT2B15 (17.6%). Molecular network and pathway analysis found enrichment related to steroid metabolic process, carboxylesterase activity, and sequence specific DNA binding. Interpretation and Conclusion. We report the presence of UGT2B gene deletion and duplication polymorphisms in Indian families. Network analysis indicates the substitutive role of other possible genes in the UGT activity. The CNVs of UGT2B genes are very common in individuals indicating that the effect is neutral in causing any suspected diseases. Avinash M. Veerappa, Prakash Padakannaya, and Nallur B. Ramachandra Copyright © 2016 Avinash M. Veerappa et al. All rights reserved. Microarrays as Model Biosensor Platforms to Investigate the Structure and Affinity of Aptamers Thu, 03 Mar 2016 09:11:00 +0000 Immobilization of nucleic acid aptamer recognition elements selected free in solution onto the surface of biosensor platforms has proven challenging. This study investigated the binding of multiple aptamer/target pairs immobilized on a commercially available microarray as a model system mimicking biosensor applications. The results indicate a minimum distance (linker length) from the surface and thymine nucleobase linker provides reproducible binding across varying conditions. An indirect labeling method, where the target was labeled with a biotin followed by a brief Cy3-streptavidin incubation, provided a higher signal-to-noise ratio and over two orders of magnitude improvement in limit of detection, compared to direct Cy3-protein labeling. We also showed that the affinities of the aptamer/target interaction can change between direct and indirect labeling and conditions to optimize for the highest fluorescence intensity will increase the sensitivity of the assay but will not change the overall affinity. Additionally, some sequences which did not initially bind demonstrated binding when conditions were optimized. These results, in combination with studies demonstrating enhanced binding in nonselection buffers, provided insights into the structure and affinity of aptamers critical for biosensor applications and allowed for generalizations in starting conditions for researchers wishing to investigate aptamers on a microarray surface. Jennifer A. Martin, Yaroslav Chushak, Jorge L. Chávez, Joshua A. Hagen, and Nancy Kelley-Loughnane Copyright © 2016 Jennifer A. Martin et al. All rights reserved. miRNA Influences in NRF2 Pathway Interactions within Cancer Models Sun, 09 Aug 2015 14:00:39 +0000 The NRF2 transcription factor (nuclear factor-erythroid 2 p45-related factor 2) has been identified as a key molecular player in orchestrating adaptive cellular interactions following a wide spectrum of cellular stress conditions that could be either extracellular or intracellular. Dysregulation of the NRF2 system is implicated in various disease states, including inflammatory conditions. The NRF2 transcription factor is also known to permit cross talk with several other essential cellular signaling pathways. Recent literature has also elucidated the potential influences of miRNA activity over modulations of the NRF2 signalling network. Consequently, further delving into the knowledge regarding the extent of miRNA-induced epigenetic gene regulatory control on key elements of the NRF2 signalling pathway and its cross talk, particularly within the context of cancer models, can prove to be of high clinical importance. This is so since such miRNAs, once identified and validated, can be potentially exploited as novel drug targets for emerging translational medicine-based therapies. Duncan Ayers, Byron Baron, and Therese Hunter Copyright © 2015 Duncan Ayers et al. All rights reserved. The Globular State of the Single-Stranded RNA: Effect of the Secondary Structure Rearrangements Sun, 09 Aug 2015 11:50:13 +0000 The mutual influence of the slow rearrangements of secondary structure and fast collapse of the long single-stranded RNA (ssRNA) in approximation of coarse-grained model is studied with analytic calculations. It is assumed that the characteristic time of the secondary structure rearrangement is much longer than that for the formation of the tertiary structure. A nonequilibrium phase transition of the 2nd order has been observed. Zareh A. Grigoryan and Armen T. Karapetian Copyright © 2015 Zareh A. Grigoryan and Armen T. Karapetian. All rights reserved. Insects’ RNA Profiling Reveals Absence of “Hidden Break” in 28S Ribosomal RNA Molecule of Onion Thrips, Thrips tabaci Thu, 12 Feb 2015 10:49:10 +0000 With an exception of aphids, insects’ 28S rRNA is thought to harbor a “hidden break” which cleaves under denaturing conditions to comigrate with 18S rRNA band to exhibit a degraded appearance on native agarose gels. The degraded appearance confounds determination of RNA integrity in laboratories that rely on gel electrophoresis. To provide guidelines for RNA profiles, RNA from five major insect orders, namely, Diptera, Hemiptera, Thysanoptera, Hymenoptera, and Lepidoptera, was compared under denaturing and nondenaturing conditions. This study confirmed that although present in most of insect’s RNA, the “hidden break” is absent in the 28S rRNA of onion thrips, Thrips tabaci. On the other hand, presence of “hidden break” was depicted in whiteflies’ 28S rRNA despite their evolutionary grouping under same order with aphids. Divergence of 28S rRNA sequences confirms variation of both size and composition of gap region among insect species. However, phylogeny reconstruction does not support speciation as a possible source of the hidden break in insect’s 28S rRNA. In conclusion, we show that RNA from a given insect order does not conform to a particular banding profile and therefore this approach cannot be reliably used to characterize newly discovered species. Rosaline Wanjiru Macharia, Fidelis Levi Ombura, and Erick Onyango Aroko Copyright © 2015 Rosaline Wanjiru Macharia et al. All rights reserved. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element against Clostridium difficile Toxin B and Sensitive Detection in Human Fecal Matter Thu, 05 Feb 2015 09:47:59 +0000 Toxin B is one of the major virulence factors of Clostridium difficile, a bacterium that is responsible for a significant number of diarrhea cases in acute care settings. Due to the prevalence of C. difficile induced diarrhea, rapid and correct diagnosis is crucial in the disease management. In this study, we have employed a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for toxin B. At the end of the 12-round selection, one MRE with high affinity  nM) for toxin B was identified. The selected MRE demonstrated low cross binding activities on negative targets: bovine serum albumin, Staphylococcus aureus alpha toxin, Pseudomonas aeruginosa exotoxin A, and cholera toxin of Vibrio cholera. A modified sandwich ELISA assay was developed utilizing the selected ssDNA MRE as the antigen capturing element and achieved a sensitive detection of 50 nM of toxin B in human fecal preparations. Ka Lok Hong, Eamonn Maher, Ryan M. Williams, and Letha J. Sooter Copyright © 2015 Ka Lok Hong et al. All rights reserved. Erratum to “Analysis of Nucleotide Sequences of the 16S rRNA Gene of Novel Escherichia coli Strains Isolated from Feces of Human and Bali Cattle” Mon, 29 Dec 2014 00:10:03 +0000 I Wayan Suardana Copyright © 2014 I Wayan Suardana. All rights reserved. Effects of Stability of Base Pairs Containing an Oxazolone on DNA Elongation Sun, 07 Dec 2014 08:34:00 +0000 The nucleoside 2,2,4-triamino-5(2H)-oxazolone (Oz) can result from oxidative damage to guanine residues in DNA. Despite differences among the three polymerases (Pol β, KF exo−, and Pol η) regarding nucleotide incorporation patterns opposite Oz, all three polymerases can incorporate guanine opposite Oz. Based on ab initio calculations, we proposed a structure for a stable Oz:G base pair. Here, to assess the stability of each Oz-containing base pair (Oz:G, Oz:A, Oz:C, and Oz:T) upon DNA replication, we determined the efficiency of Pol β-, KF exo−-, or Pol η-catalyzed primer extension beyond each base pair. With each polymerase, extension beyond Oz:G was more efficient than that beyond Oz:A, Oz:C, or Oz:T. Moreover, thermal denaturation studies revealed that the value for the duplex containing Oz:G was significantly higher than those obtained for duplexes containing Oz:A, Oz:C, or Oz:T. Therefore, the results from ab initio calculations along with those from DNA replication assays and thermal denaturation experiments supported the conclusion that Oz:G is the most stable of the Oz-containing base pairs. Masayo Suzuki, Kazuya Ohtsuki, Katsuhito Kino, Teruhiko Kobayashi, Masayuki Morikawa, Takanobu Kobayashi, and Hiroshi Miyazawa Copyright © 2014 Masayo Suzuki et al. All rights reserved. In Vitro Selection of a Single-Stranded DNA Molecular Recognition Element Specific for Bromacil Thu, 23 Oct 2014 06:15:14 +0000 Bromacil is a widely used herbicide that is known to contaminate environmental systems. Due to the hazards it presents and inefficient detection methods, it is necessary to create a rapid and efficient sensing device. Towards this end, we have utilized a stringent in vitro selection method to identify single-stranded DNA molecular recognition elements (MRE) specific for bromacil. We have identified one MRE with high affinity ( nM) and specificity for bromacil compared to negative targets of selection and other pesticides. The selected ssDNA MRE will be useful as the sensing element in a field-deployable bromacil detection device. Ryan M. Williams, Amanda R. Kulick, Srilakshmi Yedlapalli, Louisa Battistella, Cyrus J. Hajiran, and Letha J. Sooter Copyright © 2014 Ryan M. Williams et al. All rights reserved. Analysis of Nucleotide Sequences of the 16S rRNA Gene of Novel Escherichia coli Strains Isolated from Feces of Human and Bali Cattle Tue, 09 Sep 2014 00:00:00 +0000 Livestock especially cattle are known as a main reservoir of Escherichia coli O157:H7. This bacterium is considered as a pathogenic agent characterized by producing toxins, which are familiarly known as Shiga-like toxin-1 (Stx1) and Stx2. The aim of this work was to analyse the novel sequence of the 16S rRNA gene of strains isolated in this study in order to know the phylogenetic relationships between these sequences and those between the sequences of bacteria available in databanks. The results of this analysis showed that the strains KL-48(2) and SM25(1) that originated from human and cattle feces, respectively, are closely related among them and with respect to E. coli EDL 933, E. coli Sakai, E. coli ATCC 43894, E. coli O111:H-, E. coli O121:H19, E. coli O104:H4, and Shigella sonnei with more than 99% similarity values. I Wayan Suardana Copyright © 2014 I Wayan Suardana. All rights reserved. Discovery of Novel Leaf Rust Responsive microRNAs in Wheat and Prediction of Their Target Genes Tue, 12 Aug 2014 00:00:00 +0000 MicroRNAs are endogenous small noncoding RNAs which play critical roles in gene regulation. Few wheat (Triticum aestivum L.) miRNA sequences are available in miRBase repertoire and knowledge of their biological functions related to biotic stress is limited. We identified 52 miRNAs, belonging to 19 families, from next-generation transcriptome sequence data based on homology search. One wheat specific novel miRNA was identified but could not be ascribed or assigned to any known miRNA family. Differentially expressed 22 miRNAs were found between susceptible and resistant wheat near-isogenic lines inoculated with leaf rust pathogen Puccinia triticina and compared with mock inoculated controls. Most miRNAs were more upregulated in susceptible NIL compared to resistant NIL. We identified 1306 potential target genes for these 52 miRNAs with vital roles in response to stimuli, signaling, and diverse metabolic and cellular processes. Gene ontology analysis showed 66, 20, and 35 target genes to be categorized into biological process, molecular function, and cellular component, respectively. A miRNA-mediated regulatory network revealed relationships among the components of the targetome. The present study provides insight into potential miRNAs with probable roles in leaf rust pathogenesis and their target genes in wheat which establish a foundation for future studies. Dhananjay Kumar, Dharmendra Singh, Pulkit Kanodia, Kumble Vinod Prabhu, Manish Kumar, and Kunal Mukhopadhyay Copyright © 2014 Dhananjay Kumar et al. All rights reserved. Selective Evolution of Ligands by Exponential Enrichment to Identify RNA Aptamers against Shiga Toxins Tue, 15 Apr 2014 14:18:04 +0000 Infection with Shiga toxin- (Stx-) producing E. coli causes life threatening hemolytic uremic syndrome (HUS), a leading cause of acute renal failure in children. Of the two antigenically distinct toxins, Stx1 and Stx2, Stx2 is more firmly linked with the development of HUS. In the present study, selective evolution of ligands by exponential enrichment (SELEX) was used in an attempt to identify RNA aptamers against Stx1 and Stx2. After 5 rounds of selection, significant enrichment of aptamer pool was obtained against Stx2, but not against Stx1, using a RNA aptamer library containing 56 random nucleotides (N56). Characterization of individual aptamer sequences revealed that six unique RNA aptamers (mA/pC, mB/pA, mC, mD, pB, and pD) recognized Stx2 in a filter binding assay. None of these aptamers bound Stx1. Aptamers mA/pC, mB/pA, mC, and mD, but not pB and pD, partially blocked binding of Alexa 488-labeled Stx2 with HeLa cells in a flow cytometry assay. However, none of the aptamers neutralized Stx2-mediated cytotoxicity and death of HeLa cells. Sreerupa Challa, Saul Tzipori, and Abhineet Sheoran Copyright © 2014 Sreerupa Challa et al. All rights reserved. Expression Analysis of Sugarcane Aquaporin Genes under Water Deficit Sun, 29 Dec 2013 11:51:41 +0000 The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific. Manassés Daniel da Silva, Roberta Lane de Oliveira Silva, José Ribamar Costa Ferreira Neto, Ana Carolina Ribeiro Guimarães, Daniela Truffi Veiga, Sabrina Moutinho Chabregas, William Lee Burnquist, Günter Kahl, Ana Maria Benko-Iseppon, and Ederson Akio Kido Copyright © 2013 Manassés Daniel da Silva et al. All rights reserved. Multipyrene Tandem Probes for Point Mutations Detection in DNA Tue, 24 Dec 2013 08:12:21 +0000 Here we report design, synthesis and characterization of highly sensitive, specific and stable in biological systems fluorescent probes for point mutation detection in DNA. The tandems of 3′- and 5′-mono- and bis-pyrene conjugated oligo(2′-O-methylribonucleotides), protected by 3′-“inverted” thymidine, were constructed and their potential as new instruments for genetic diagnostics was studied. Novel probes have been shown to exhibit an ability to form stable duplexes with DNA target due to the stabilizing effect of multiple pyrene units at the junction. The relationship between fluorescent properties of developed probes, the number of pyrene residues at the tandem junction, and the location of point mutation has been studied. On the basis of the data obtained, we have chosen the probes possessing the highest fluorescence intensity along with the best mismatch discrimination and deletion and insertion detection ability. Application of developed probes for detection of polymorphism C677T in MTHFR gene has been demonstrated on model systems. Svetlana A. Kholodar, Darya S. Novopashina, Mariya I. Meschaninova, and Alya G. Venyaminova Copyright © 2013 Svetlana A. Kholodar et al. All rights reserved. Transcriptionally Repressive Chromatin Remodelling and CpG Methylation in the Presence of Expanded CTG-Repeats at the DM1 Locus Mon, 23 Dec 2013 15:28:42 +0000 An expanded CTG-repeat in the 3′ UTR of the DMPK gene is responsible for myotonic dystrophy type I (DM1). Somatic and intergenerational instability cause the disease to become more severe during life and in subsequent generations. Evidence is accumulating that trinucleotide repeat instability and disease progression involve aberrant chromatin dynamics. We explored the chromatin environment in relation to expanded CTG-repeat tracts in hearts from transgenic mice carrying the DM1 locus with different repeat lengths. Using bisulfite sequencing we detected abundant CpG methylation in the regions flanking the expanded CTG-repeat. CpG methylation was postulated to affect CTCF binding but we found that CTCF binding is not affected by CTG-repeat length in our transgenic mice. We detected significantly decreased DMPK sense and SIX5 transcript expression levels in mice with expanded CTG-repeats. Expression of the DM1 antisense transcript was barely affected by CTG-repeat expansion. In line with altered gene expression, ChIP studies revealed a locally less active chromatin conformation around the expanded CTG-repeat, namely, decreased enrichment of active histone mark H3K9/14Ac and increased H3K9Me3 enrichment (repressive chromatin mark). We also observed binding of PCNA around the repeats, a candidate that could launch chromatin remodelling cascades at expanded repeats, ultimately affecting gene transcription and repeat instability. Judith Rixt Brouwer, Aline Huguet, Annie Nicole, Arnold Munnich, and Geneviève Gourdon Copyright © 2013 Judith Rixt Brouwer et al. All rights reserved. Comparative (Computational) Analysis of the DNA Methylation Status of Trinucleotide Repeat Expansion Diseases Mon, 23 Dec 2013 09:51:50 +0000 Previous studies have examined DNA methylation in different trinucleotide repeat diseases. We have combined this data and used a pattern searching algorithm to identify motifs in the DNA surrounding aberrantly methylated CpGs found in the DNA of patients with one of the three trinucleotide repeat (TNR) expansion diseases: fragile X syndrome (FRAXA), myotonic dystrophy type I (DM1), or Friedreich’s ataxia (FRDA). We examined sequences surrounding both the variably methylated (VM) CpGs, which are hypermethylated in patients compared with unaffected controls, and the nonvariably methylated CpGs which remain either always methylated (AM) or never methylated (NM) in both patients and controls. Using the J48 algorithm of WEKA analysis, we identified that two patterns are all that is necessary to classify our three regions CCGG* which is found in VM and not in AM regions and AATT* which distinguished between NM and VM + AM using proportional frequency. Furthermore, comparing our software with MEME software, we have demonstrated that our software identifies more patterns than MEME in these short DNA sequences. Thus, we present evidence that the DNA sequence surrounding CpG can influence its susceptibility to be de novo methylated in a disease state associated with a trinucleotide repeat. Mohammadmersad Ghorbani, Simon J. E. Taylor, Mark A. Pook, and Annette Payne Copyright © 2013 Mohammadmersad Ghorbani et al. All rights reserved. Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis Tue, 17 Dec 2013 10:36:09 +0000 Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations. John P. Jakupciak, Jeffrey M. Wells, Richard J. Karalus, David R. Pawlowski, Jeffrey S. Lin, and Andrew B. Feldman Copyright © 2013 John P. Jakupciak et al. All rights reserved. Oligonucleotide-Based Therapy for FTD/ALS Caused by the C9orf72 Repeat Expansion: A Perspective Sun, 17 Nov 2013 10:51:58 +0000 Amyotrophic lateral sclerosis (ALS) is a progressive and lethal disease of motor neuron degeneration, leading to paralysis of voluntary muscles and death by respiratory failure within five years of onset. Frontotemporal dementia (FTD) is characterised by degeneration of frontal and temporal lobes, leading to changes in personality, behaviour, and language, culminating in death within 5–10 years. Both of these diseases form a clinical, pathological, and genetic continuum of diseases, and this link has become clearer recently with the discovery of a hexanucleotide repeat expansion in the C9orf72 gene that causes the FTD/ALS spectrum, that is, c9FTD/ALS. Two basic mechanisms have been proposed as being potentially responsible for c9FTD/ALS: loss-of-function of the protein encoded by this gene (associated with aberrant DNA methylation) and gain of function through the formation of RNA foci or protein aggregates. These diseases currently lack any cure or effective treatment. Antisense oligonucleotides (ASOs) are modified nucleic acids that are able to silence targeted mRNAs or perform splice modulation, and the fact that they have proved efficient in repeat expansion diseases including myotonic dystrophy type 1 makes them ideal candidates for c9FTD/ALS therapy. Here, we discuss potential mechanisms and challenges for developing oligonucleotide-based therapy for c9FTD/ALS. Stephanie A. Fernandes, Andrew G. L. Douglas, Miguel A. Varela, Matthew J. A. Wood, and Yoshitsugu Aoki Copyright © 2013 Stephanie A. Fernandes et al. All rights reserved. Immobilization of DNA Aptamers on Polyester Cloth for Antigen Detection by Dot Blot Immunoenzymatic Assay (Aptablot) Wed, 30 Oct 2013 11:20:57 +0000 A simple dot blot immunoenzymatic assay system was developed using polyester cloth coated with an oligo-DNA aptamer to provide a high-affinity macroporous surface for the efficient capture of a model protein analyte (thrombin) in complex sample matrices such as foods. Bound thrombin was detected immunoenzymatically using a peroxidase-linked antithrombin antibody and a chromogenic substrate. A unique feature of this approach, which we have termed “aptablot,” is the facile immobilization of DNA aptamers on the polyester surface by cross-linking with a brief exposure to ultraviolet light, and the simple assay format obviating the need for specialized instruments. The assay principle described herein should be broadly applicable to many situations where analytes must be detected in complex samples, with the main limiting factor being the availability of suitable DNA aptamers. Sally Smiley, Maria DeRosa, and Burton Blais Copyright © 2013 Sally Smiley et al. All rights reserved. Simultaneous Detection of Different MicroRNA Types Using the ZIP-Code Array System Mon, 02 Sep 2013 08:16:18 +0000 MicroRNAs (miRNAs) are important negative regulators of gene expression. Their implication in tumorigenesis is based on their dysregulation in many human cancer diseases. Interestingly, in tumor cells, an altered ratio of precursor and mature miRNA levels has been described. Consequently, differences in miRNA type levels have a high potential as biomarkers and comparative high-throughput-based detection might permit a more accurate characterization of subtypes, especially in the case of very heterogeneous tumor entities. Several molecular methods exist for the detection of mature and precursor miRNAs. DNA microarrays are predestinated as a high-throughput method for comprehensive miRNA detection in tumors. However, the simultaneous array-based detection of both these miRNA types is limited because the mature miRNA sequence is identically present in both forms. Here we present a ZIP-code DNA microarray-based system in combination with a novel labeling approach, which enables the simultaneous detection of precursor and mature miRNAs in one single experiment. Using synthetic miRNA templates, we demonstrate the specificity of the method for the different miRNA types, as well as the detection range up to four orders of magnitude. Moreover, mature and precursor miRNAs were detected and validated in human tumor cells. Sonja U. Weishaupt, Steffen Rupp, and Karin Lemuth Copyright © 2013 Sonja U. Weishaupt et al. All rights reserved. R-Loop Formation In Trans at an AGGAG Repeat Mon, 26 Aug 2013 08:34:06 +0000 Formation of RNA-DNA hybrid, or R-loop, was studied in vitro by transcribing an AGGAG repeat with T7 RNA polymerase. When ribonuclease T1 was present, R-loop formation in cis was diminished, indicating that the transcript was separated from the template and reassociated with it. The transcript was found to form an R-loop in trans with DNA comprising the AGGAG repeat, when the DNA was supercoiled. Results of chemical modification indicated that the duplex opened at the AGGAG repeat under negative supercoiling. Kazuya Toriumi, Takuma Tsukahara, and Ryo Hanai Copyright © 2013 Kazuya Toriumi et al. All rights reserved. Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplification during PCR Sun, 21 Jul 2013 13:34:28 +0000 Thermus thermophilus MutS, a thermostable mismatch-recognizing protein, is utilized in PCR to suppress nonspecific amplification by preventing synthesis from mismatched primers. T. thermophilus RecA also decreases nonspecific amplification by promoting proper hybridization between the primer and template. We observed that MutS and RecA function under the same reaction conditions and that MutS and RecA do not preclude each other. Furthermore, there were some DNA sequences for which only one of the 2 proteins effectively suppressed nonspecific amplification. The simultaneous use of MutS and RecA is a more attractive error-suppressing technique than the use of either of the 2 proteins alone. Kenji Fukui and Seiki Kuramitsu Copyright © 2013 Kenji Fukui and Seiki Kuramitsu. All rights reserved. Mesenchymal Stem Cell Therapy in Diabetes Mellitus: Progress and Challenges Wed, 15 May 2013 07:57:14 +0000 Advanced type 2 diabetes mellitus is associated with significant morbidity and mortality due to cardiovascular, nervous, and renal complications. Attempts to cure diabetes mellitus using islet transplantation have been successful in providing a source for insulin secreting cells. However, limited donors, graft rejection, the need for continued immune suppression, and exhaustion of the donor cell pool prompted the search for a more sustained source of insulin secreting cells. Stem cell therapy is a promising alternative for islet transplantation in type 2 diabetic patients who fail to control hyperglycemia even with insulin injection. Autologous stem cell transplantation may provide the best outcome for those patients, since autologous cells are readily available and do not entail prolonged hospital stays or sustained immunotoxic therapy. Among autologous adult stem cells, mesenchymal stem cells (MSCs) therapy has been applied with varying degrees of success in both animal models and in clinical trials. This review will focus on the advantages of MSCs over other types of stem cells and the possible mechanisms by which MSCs transplant restores normoglycemia in type 2 diabetic patients. Sources of MSCs including autologous cells from diabetic patients and the use of various differentiation protocols in relation to best transplant outcome will be discussed. Nagwa El-Badri and Mohamed A. Ghoneim Copyright © 2013 Nagwa El-Badri and Mohamed A. Ghoneim. All rights reserved. Thermodynamic and Structural Analysis of DNA Damage Architectures Related to Replication Sun, 28 Apr 2013 10:35:00 +0000 Damaged DNA, generated by the abstraction of one of five hydrogen atoms from the 2′-deoxyribose ring of the nucleic acid, can contain a variety of lesions, some of which compromise physiological processes. Recently, DNA damage, resulting from the formation of a C3′-thymidinyl radical in DNA oligomers, was found to be dependent on nucleic acid structure. Architectures relevant to DNA replication were observed to generate larger amounts of strand-break and 1-(2′-deoxy-β-D-threo-pentofuranosyl)thymidine formation than that observed for duplex DNA. To understand how this damage can affect the integrity of DNA, the impact of C3′-thymidinyl radical derived lesions on DNA stability and structure was characterized using biophysical methods. DNA architectures evaluated include duplex DNA (dsDNA), single 3′ or 5′-overhangs (OvHgs), and forks. Thermal melting analysis and differential scanning calorimetry measurements indicate that an individual 3′-OvHg is more destabilizing than a 5′-OvHg. The presence of a terminal 3′ or 5′ phosphate decreases the to the same extent, while the effect of the phosphate at the ss-dsDNA junction of OvHgs is dependent on sequence. Additionally, the effect of 1-(2′-deoxy-β-D-threo-pentofuranosyl)thymidine is found to depend on DNA architecture and proximity to the 3′ end of the damaged strand. Nicholas J. Amato, Christopher N. Mwai, Timothy C. Mueser, and Amanda C. Bryant-Friedrich Copyright © 2013 Nicholas J. Amato et al. All rights reserved.