Quadruplexes of human telomere DNA analogs designed to contain G:A:G:A,G:G:A:A,and A:A:A:A tetrads

Replacement of two to four guanines by adenines in the human telomere DNA repeat dG₃(TTAG₃)₃ did not hinder the formation of quadruplexes if the substitutions took place in the terminal tetrad bridged by the diagonal loop of the intramolecular antiparallel three‐tetrad scaffold, as proved by CD and PAGE in both Na⁺ and K⁺ solutions. Thermodynamic data showed that, in Na⁺ solution, the dG₃(TTAG₃)₃ quadruplex was destabilized, the least by the two G:A:G:A tetrads, the most by the G:G:A:A tetrad in which the adenosines replaced syn‐guanosines. In physiological K⁺ solution, the highest destabilization was caused by the 4A tetrad. In K⁺, only the unmodified dG₃(TTAG₃)₃ quadruplex rearranged into a K⁺‐dependent quadruplex form, none of the multiple adenine‐modified structures did so. This may imply biological consequences for nonrepaired A‐for‐G mutations. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 880–886, 2010.     

Hormone interactions and regulation of PsPK2::GUS compared with DR5::GUS and PID::GUS in Arabidopsis thaliana

The putative pea PINOID homolog, PsPK2, is expressed in all growing plant parts and is positively regulated by auxin, gibberellin, and cytokinin. Here, we studied hormonal regulation of PsPK2::GUS expression compared with DR5::GUS and PID::GUS in Arabidopsis. PsPK2::GUS, DR5::GUS, and PID::GUS expression in Arabidopsis shoots is mainly localized in the stipules, hydathodes, veins, developing leaves, and cotyledons. Unlike DR5::GUS, PsPK2::GUS, and PID::GUS are weakly expressed in root tips. Both DR5::GUS and PsPK2::GUS are induced by different auxins and are more sensitive to methyl indole acetic acid, 4-chloro-indole acetic acid, and α-naphthalene acetic acid than others. GA(3) has no significant effect on GUS activity in DR5::GUS-transformed seedlings compared to the control, but induction by auxin and gibberellin in combination is synergistic. Cytokinin increases auxin transport in Arabidopsis seedlings. Auxin, gibberellin, and cytokinin all increase GUS activity in shoots of PsPK2::GUS transformed plants compared to the control. However, only auxin and gibberellin increase GUS activity in PID::GUS shoots. In conclusion, auxin, gibberellin, and cytokinin positively regulate PsPK2 expression in shoots, but not in roots. Auxin and gibberellin also upregulate AtPIN1 and LEAFY expression, which is similar to PsPIN1 and Uni in pea. With minor exceptions, the orthologous genes from both species are regulated similarly.     

Experimental demonstration of T:(G:G:G:G):T hexad and T:A:A:T tetrad alignments within a DNA quadruplex stem

A template-based approach was used to design unprecedented architectural motifs into a known DNA framework. The structure formed by the sequence d(GCGGTTGGAT) in 0.1 M Na(+) solution has been determined using molecular dynamics simulations constrained by distance and dihedral restraints derived from NMR experiments. The molecular topology has been previously observed for the sequence d(GCGGTGGAT) (Webba da Silva, M. (2003) Biochemistry 42, 14356-65). Insertion of a single thymine into the double chain reversal formed by the segment GGTGG results in the unprecedented experimental demonstration of a T:(G:G:G:G):T hexad. The bi-stranded hexad results from the pairing alignment of two G(T-G) triads. Each triad results from recognition of the sheared edge of a guanine by the Watson-Crick edge of a thymine of the segment GGTTGG. The alignment is stabilized by base-stacking of the thymine to the sugar pucker of the preceding thymine. The latter is involved in formation of the T:A:A:T tetrad alignment by forming a hydrogen bond with the free amino proton of a Watson-Crick aligned A:A mispair. We have thus established that residues in double chain reversal loops linking juxtaposed tetrads of a quadruplex stem may facilitate formation of yet unknown hydrogen bond alignments. By employing a systematic approach analysis of sequence motifs appearing in double chain reversals, bridging tetrad layers should allow for the prediction of topologies and architectural motifs appearing in biologically relevant genomic regions.     

Oka: Behind the Barricades: Kanehsatake: 270 Years of Resistance: Spudwrench: Kahnawake Man: My Name is Kahentiiosta: Acts of Defiance

Oka: Behind the Barricades. 1998. 312 minutes. For more information contact the National Film Board of Canada, 3155 Côte de Lisse Rd., St. Laurent, Quebec H4N 2N4. Series of four videos:
Kanehsatake: 270 Years of Resistance. 1993. 120 minutes. Directed by Alanis Obomsawin .
Spudwrench: Kahnawake Man. 1997. 58 minutes. Directed by Alanis Obomsawin .
My Name is Kahentiiosta. 1995. 30 minutes. Directed by Alanis Obomsawin .
Acts of Defiance. 1992. 104 minutes. Directed by Alec MacLeod .     

A dimeric RNA quadruplex architecture comprised of two G:G(:A):G:G(:A) hexads,G:G:G:G tetrads and UUUU loops

Using CD and NMR, we determined the structure of an RNA oligomer, r(GGAGGUUUUGGAGG) (R14), comprising two GGAGG segments joined by a UUUU segment. A modified quadruplex structure was observed for r(GGAGGUUUUGGAGG) in solution even in the absence of K(+). An unusually stable dimeric RNA quadruplex architecture formed from two strands of r(GGAGGUUUUGGAGG) at low K(+) concentration is reported here. In each strand of r(GGAGGUUUUGGAGG), two sets of successive turns in the GGAGG segments and turns at both ends of the UUUU loops drive four G-G steps to align in a parallel manner, a core with two stacked G-tetrads being formed. Two adenine bases bind to two edges of one G:G:G:G tetrad through the sheared G:A mismatch augmenting the tetrad into a G:G(:A):G:G(:A) hexad. Thus, one molecule of r(GGAGGUUUUGGAGG) folds into a modified quadruplex comprising a G:G:G:G tetrad, a UUUU double-chain reversal loop and a G:G(:A):G:G(:A) hexad. Two such molecules further associate by stacking through the dimeric hexad-hexad interface with a rotational symmetry. The ribose rings of most nucleotides take S (close to C2'-endo) puckering, which is unusual for an RNA. K(+) can increase the stability of this quadruplex structure; the number of bound K(+) was estimated from the results of the titration experiment. Besides G:G and G:A mismatches, a network of hydrogen bonds including O4'-NH(2) and C-H..O hydrogen bonds, and the extensive base stacking contribute to the high thermodynamic stability of R14. Our results could provide the stereochemical and thermodynamic basis for elucidating the biological role of the GGAGG-containing RNA segments abundantly existing in various RNAs. Relevance to quadruplex-mediated mRNA-FMRP binding and HIV-1 genome RNA dimerization is discussed.     

Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids.

Plasmids were constructed in which DNA damage-inducible promoters recA, uvrA, and alkA from Escherichia coli were fused to the Vibrio fischeri luxCDABE operon. Introduction of these plasmids into E. coli allowed the detection of a dose-dependent response to DNA-damaging agents, such as mitomycin and UV irradiation. Bioluminescence was measured in real time over extended periods. The fusion of the recA promoter to luxCDABE showed the most dramatic and sensitive responses. lexA dependence of the bioluminescent SOS response was demonstrated, confirming that this biosensor's reports were transmitted by the expected regulatory circuitry. Comparisons were made between luxCDABE and lacZ fusions to each promoter. It is suggested that the lux biosensors may have use in monitoring chemical, physical, and genotoxic agents as well as in further characterizing the mechanisms of DNA repair.     

Focus: Rare Disease: Chronic Pulmonary Aspergillosis: A Brief Review

Chronic Pulmonary Aspergillosis (CPA) is a destructive pulmonary disease caused by a fungal infection, affecting mainly individuals with prior or concurrent pulmonary conditions. It has a global prevalence of 42 per 100,000 population, but in the US and Europe, prevalence is less than 1 per 100,000. The clinical definition of CPA is based on various factors accounting for comorbidities, clinical presentation, and duration. It may be categorized into five subtypes that the disease may evolve between over time. Based on global consensus covering the spectrum of low-resource to high-resource settings, diagnosis is a multi-factorial process that involves a combination of clinical presentation persisting over 3 months, radiological findings, positive culture growth, and serological tests. CPA remains underdiagnosed due to a lack of awareness and is often misdiagnosed due to the comorbidities present. Treatment options are limited due to a lack of research. Furthermore, associated comorbidities and drug interactions further complicate treatment plans. Follow-up throughout treatment should be based on understanding the predictors of mortality. Identification of potential relapse or resistance to antifungal therapy is crucial to limit the low long-term survival rate. Awareness surrounding this devastating disease needs to be raised further to enable earlier identification, improve understanding of patient factors associated with prognosis, and the future potential for targeted therapies. This review aims to raise awareness of this rare condition among practitioners, by providing an overview of common risk factors influencing the prevalence and incidence of the disease. We further discuss current approaches and recent advancements in CPA diagnosis and treatment.     

Intramolecular higher order packing of parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad of GGA triplet repeat DNA

GGA triplet repeats are widely dispersed throughout eukaryotic genomes and are frequently located within biologically important regions such as gene regulatory regions and recombination hot spot sites. We determined the structure of d(GGA)4 (12-mer) under physiological conditions and founded the formation of an intramolecular parallel quadruplex for the first time. Later, a similar architecture to that of the intramolecular parallel quadruplex was found for a telomere DNA in the crystalline state. Here, we have determined the structure of d(GGA)8 (24-mer) under physiological conditions. Two intramolecular parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad are formed in d(GGA)8. These quadruplexes are packed in a tail-to-tail manner. This is the first demonstration of the intramolecular higher order packing of quadruplexes at atomic resolution. K+ ions, but not Na+ ones, are critically required for the formation of this unique structure. The elucidated structure suggests the mechanisms underlying the biological events related to the GGA triplet repeat. Furthermore, in the light of the structure, the mode of the higher order packing of the telomere DNA is discussed.     

Assessment of heavy metal bioavailability using Escherichia coli zntAp::lux and copAp::lux-based biosensors

To determine the amount of metals detectable by bacteria, two plasmids were constructed in which the metal-inducible zntA and copA promoters from Escherichia coli were fused to a promoterless Vibrio fischeri luxCDABE operon. The luminescence response of E. coli bearing these constructs was studied as a function of the concentration of several heavy metals and was shown to be influenced by cell growth phase. The zntAp::lux fusion is induced mainly by salts of cadmium, lead, mercury and zinc, with significant induction by other metal ions, whereas the specificity of copA induction is restricted to copper and silver. In optimized assay conditions, metals could be detected at threshold concentrations ranging from nanomolar to micromolar, with maximal induction observed after only 60-100 min incubation. The ability of these biosensor strains to distinguish bioavailable quantities of metals in a sample makes them good candidates as useful tools to monitor metal contamination in environmental samples.     

Phylogeny of Bembidion and related ground beetles (Coleoptera: Carabidae: Trechinae: Bembidiini: Bembidiina)

The phylogeny of the large genus Bembidion and related genera is inferred from four nuclear protein-coding genes (CAD, wingless, arginine kinase, and topoisomerase I), ribosomal DNA (28S and 18S), and the mitochondrial gene cytochrome oxidase I (COI). 230 of the more than 1200 species of Bembidion are sampled, as well as 26 species of five related genera, and 14 outgroups. Nuclear copies (numts) of COI were found sparsely scattered through sampled species. The resulting phylogeny, based upon individual gene analyses and combined analyses using maximum likelihood and parsimony, is very well supported at most nodes. Additional analyses explored the evidence, and corroborate the phylogeny. Seven analyses, each with one of the seven genes removed from the combined matrix, were also conducted, and yielded maximum likelihood bootstrap trees sharing over 92% of their nodes with the original, well-resolved bootstrap trees based on the complete set of seven genes. All key nodes were present in all seven analyses missing a single gene, indicating that support for these nodes comes from at least two genes. In addition, the inferred maximum likelihood tree based on the combined matrix is well-behaved and self-predicting, in that simulated evolution of sequences on the inferred tree under the inferred model of evolution yields a matrix from which all but one of the model tree's clades are recovered with bootstrap value >50, suggesting that internal branches in the tree may be of a length to yield sequences sufficient to allow their inference. All likelihood analyses were conducted under both a proportion-invariable plus gamma site-to-site rate variation model, as well as a simpler gamma model. The choice of model did not have a major effect on inferred phylogenies or their bootstrap values. The inferred phylogeny shows that Bembidarenas is not closely related to Bembidiina, and Phrypeus is likely distant as well; the remaining genera of Bembidiina form a monophyletic group. Lionepha, formerly considered a subgenus of Bembidion, is shown to be outside of the clade of Asaphidion+Bembidion, and is separated as its own genus. B. (Phyla) obtusum is quite isolated within Bembidion, and there is some evidence that the remaining Bembidion form a clade. Within Bembidion, there are three large clades that are well-supported, the Bembidion, Odontium, and Ocydromus Series. The Bembidion Series contains Bembidion (s. str.), Notaphus, Furcacampa, Emphanes, Trepanedoris, Diplocampa, and related Holarctic species; all species from South America, Australia, New Zealand; and most species from southern Africa and Madagascar. All species in South America, except for members of Notaphus and Nothocys, form a clade, the Antiperyphanes Complex, which has independently radiated into body forms and niches occupied by multiple, independent Northern-Hemisphere forms. All species from New Zealand, including Zecillenus, and Australian species formerly placed in Ananotaphus together form a clade. Bembidion (s. str.) and Cyclolopha are in a clade with the Old World, Southern Hemisphere lineages Notaphocampa, Sloanephila, and Omotaphus. The large subgenus Notaphus appears to have originated in South America, with all Northern Hemisphere Notaphus arising from within a south-temperate grade. All major variation in frontal furrows on the head is contained within the Bembidion Series. The Odontium Series contains subgenera Hirmoplataphus and Hydriomicrus, which together are the sister clade of Odontium, Bracteon, Ochthedromus, Pseudoperyphus, and Microserrullula. The very large Ocydromus Series, dominant in the Holarctic region, includes the Ocydromus Complex, with many subgenera, including Hypsipezum and Leuchydrium; the phylogeny within this group is notably at odds with the current classification. Also included in the Ocydromus Series are Nepha and Bembidionetolitzkya, as well as the Princidium Complex, in which the intertidal B. (Cillenus) laterale falls. Outside these three series are a number of smaller groups, including the Plataphus Complex (containing Blepharoplataphus, Plataphus, the latter including Plataphodes); the Hydrium Complex (Metallina, Chlorodium, and Hydrium, which contains Eurytrachelus), whose sister group might be subgenus Andrewesa; Trechonepha and Liocosmius, which might be sisters; and B. (Melomalus) planatum, which is not close to Plataphus. There is some evidence that these groups plus the Ocydromus and Odontium Series form a clade. A few enigmatic groups were harder to place. The sister group of the pair Philochthus plus Philochthemphanes might be B. wickhami; Eupetedromus is well outside the three major series and not related to Notaphus; the high-elevation Asian group Hoquedela is a very isolated lineage. Notaphiellus is removed from synonymy with Nothocys, and placed in synonymy with Notaphus; Plataphodes is synonymized with Plataphus, as Plataphus is paraphyletic otherwise; Eurytrachelus is synonymized with Hydrium. A new subgenus, Lindrochthus, is described to house the distinctive B. wickhami. The implications of the inferred phylogeny for some morphological characters used in Bembidiina systematics are explored, and some of the most widely used (e.g., location of discal seta ed3 on the elytron, and shape of the shoulder) are shown to be notably homoplastic. For example, the location of elytral seta ed3 has undergone at least nine transitions between two states.     

Focus: Microbiome: Metagenomic Assembly: Overview,Challenges and Applications

Advances in sequencing technologies have led to the increased use of high throughput sequencing in characterizing the microbial communities associated with our bodies and our environment. Critical to the analysis of the resulting data are sequence assembly algorithms able to reconstruct genes and organisms from complex mixtures. Metagenomic assembly involves new computational challenges due to the specific characteristics of the metagenomic data. In this survey, we focus on major algorithmic approaches for genome and metagenome assembly, and discuss the new challenges and opportunities afforded by this new field. We also review several applications of metagenome assembly in addressing interesting biological problems.     

Review: Gastric cancer: Basic aspects

Gastric cancer is still one of the most prevalent and deadliest cancers in the world. Although our knowledge about the disease has progressed extraordinarily, this has not been accompanied by our capacity to effectively treat the disease. In the last years, immunotherapy made its way into the cancer field and was responsible for major changes in the treatment success rates for several cancer types. Although gastric cancer was not among the first successful targets of this type of therapy, the relationship between this type of cancer, immunosurveillance and immunotherapy is now being actively researched. In this article, we review the literature of the past year regarding the relationship between gastric cancer, its immune microenvironment and response to immunotherapy. Published data indicate that the immune microenvironment influences the clinical behaviour of gastric cancer, and is correlated with its histologic and molecular subtypes with an emphasis on the microsatellite‐ and EBV‐positive tumour subgroups. Although the literature regarding response to immunotherapy is scarce, there is good evidence that patient stratification for immunotherapy is going to become a reality in gastric cancer.     

BIO::Phylo-phyloinformatic analysis using perl

Background  

Phyloinformatic analyses involve large amounts of data and metadata of complex structure. Collecting, processing, analyzing, visualizing and summarizing these data and metadata should be done in steps that can be automated and reproduced. This requires flexible, modular toolkits that can represent, manipulate and persist phylogenetic data and metadata as objects with programmable interfaces.     

Arhynchobdellida (Annelida: Oligochaeta: Hirudinida): phylogenetic relationships and evolution

A remarkable diversity of life history strategies, geographic distributions, and morphological characters provide a rich substrate for investigating the evolutionary relationships of arhynchobdellid leeches. The phylogenetic relationships, using parsimony analysis, of the order Arhynchobdellida were investigated using nuclear 18S and 28S rDNA, mitochondrial 12S rDNA, and cytochrome c oxidase subunit I sequence data, as well as 24 morphological characters. Thirty-nine arhynchobdellid species were selected to represent the seven currently recognized families. Sixteen rhynchobdellid leeches from the families Glossiphoniidae and Piscicolidae were included as outgroup taxa. Analysis of all available data resolved a single most-parsimonious tree. The cladogram conflicted with most of the traditional classification schemes of the Arhynchobdellida. Monophyly of the Erpobdelliformes and Hirudiniformes was supported, whereas the families Haemadipsidae, Haemopidae, and Hirudinidae, as well as the genera Hirudo or Aliolimnatis, were found not to be monophyletic. The results provide insight on the phylogenetic positions for the taxonomically problematic families Americobdellidae and Cylicobdellidae, the genera Semiscolex, Patagoniobdella, and Mesobdella, as well as genera traditionally classified under Hirudinidae. The evolution of dietary and habitat preferences is examined.     

Review: Gastric malignancies: Basic aspects

Gastric cancer is the third deadliest cancer in the world, and the absolute number of cases is increasing every year due to aging and growing of high‐risk populations. This disease is a consequence of the complex interaction of microbial agents, with environmental and host factors, resulting in the dysregulation of multiple oncogenic and tumor‐suppressing signaling pathways. Despite the advances in our understanding of carcinogenesis, there are still reduced therapeutic options for patients with gastric cancer. In recent years, genomic analyses of gastric tumors have emphasized their molecular heterogeneity. The distinction of gastric cancer molecular subtypes may be a key to identify novel therapeutic targets, to predict patient outcome and response to therapy, and to guide early diagnosis strategies. In this review, we summarize the most recent updates on the relationship between microbial agents and gastric cancer, in particular, Helicobacter pylori, the non‐H pylori microbiome, and Epstein‐Barr virus. We also highlight the main advances made in the past year regarding the molecular characterization of gastric cancer, especially the signatures with potential clinical utility.