Arrayed primer extension: solid-phase four-color DNA resequencing and mutation detection technology

The technology and application of arrayed primer extension (APEX) is presented. We describe an integrated system with DNA chip and template preparation, multiplex primer extension on the array, fluorescence imaging, and data analysis. The method is based upon an array of oligonucleotides, immobilized via the 5' end on a glass surface. A patient DNA is amplified by PCR, digested enzymatically, and annealed to the immobilized primers, which promote sites for template-dependent DNA polymerase extension reactions using four unique fluorescently labeled dideoxy nucleotides. A mutation is detected by a change in the color code of the primer sites. The technology was applied to the analysis of 10 common beta-thalassemia mutations. Nine patient DNA samples, each of which carries a different mutation, and four wild-type DNA samples were correctly identified. The signal-to-noise ratio of this technology is, on the average, 40:1, which enables the identification of heterozygous mutations with a high confidence level. The APEX method can be applied to any DNA target for efficient analysis of mutations and polymorphisms.     

Body movements and their role as triggers of heartbeats in pupae of the Colorado potato beetle Leptinotarsa decemlineata

Rhythmic body movements and their role as triggers of intermittent heartbeats were studied in pupae of Leptinotarsa decemlineata Say. Heartbeats and body movements were recorded simultaneously by means of an optical method (infra‐red cardiography) combined with respirometry. IR‐cardiography allows heartbeats and body movements to be distinguished on the basis of their different rates (40–80 min^?1 and 4–8 min^?1, respectively) and amplitude. In the mid‐interecdysial period, abdominal movements in the pupae were always accompanied by heart activity beginning after the first 5–12 abdominal strokes. Simultaneous periods of abdominal movements and heartbeats lasted 2–5 min, while the intervening pause ranged from 40 to 72 min at 24°C. Experiments of forced heart activity showed that a slight external tactile stimulus (prodding once with a single hair), applied prior to an expected heartbeat bout, evoked abdominal movements followed soon afterwards by contractions of the dorsal vessel. Repeated prodding with a hair evoked body rotating movements (1–3 strokes) at any time with heartbeats starting at the first movement. We surmise that rhythmic body movements play an active role in the triggering of heart activity in pupae according to the principle of cardiac reflex response. This is a possible mechanism for synchronizing periods of heart activity with periods of rhythmic body movements. Haemolymph circulation in pupae is favoured when heartbeat bouts are accompanied by active body movements.     

Trapping the Pasture Odorscape Using Open-Air Solid-Phase Micro Extraction,a Tool to Assess Grassland Value

Besides supporting cattle feeding, grasslands are home to a diversity of plants and insects that interact with each other by emitting volatile compounds. The aim of this work was to develop a method to determine permanent grassland odorscape and relate it to flower-visiting insects. Two grasslands were chosen for their contrasting levels of botanical diversity, resulting from differing grazing managements. Measurements were made over two periods of three consecutive days at the beginning of grazing, and just after the cows had left the plots. Volatile compounds were trapped using solid-phase microextraction (SPME) fibers exposed eight hours a day in three exclosures per plot, and then analyzed by gas-chromatography-mass spectrometry (GC-MS). Insects were trapped using pan traps and a net, sorted and counted. The open air SPME method yielded volatile compound profiles that were richer than maize field profiles, comprising the common green leaf volatiles (GLV) and more specific ones. Differences between the odorscapes of the two grasslands were found, but they were not as marked as expected from their botanical composition. By contrast, there were sharp differences between the two periods, resulting from the combined effects of changes in weather conditions, plant phenological stage and grazing progress. Several correlations between insect counts and volatile compounds were found. Although their correlation coefficients were low, some of them were confirmed when tested by Spearman rank correlation, and could be logically explained. This method of grassland odorscape deserves to be developed because it can provide information on many aspects of grassland function and on the stresses that grassland plants undergo.     

Engagement of the ATR-Dependent DNA Damage Response at the Human Papillomavirus 18 Replication Centers during the Initial Amplification

We have previously demonstrated that the human papillomavirus (HPV) genome replicates effectively in U2OS cells after transfection using electroporation. The transient extrachromosomal replication, stable maintenance, and late amplification of the viral genome could be studied for high- and low-risk mucosal and cutaneous papillomaviruses. Recent findings indicate that the cellular DNA damage response (DDR) is activated during the HPV life cycle and that the viral replication protein E1 might play a role in this process. We used a U2OS cell-based system to study E1-dependent DDR activation and the involvement of these pathways in viral transient replication. We demonstrated that the E1 protein could cause double-strand DNA breaks in the host genome by directly interacting with DNA. This activity leads to the induction of an ATM-dependent signaling cascade and cell cycle arrest in the S and G₂ phases. However, the transient replication of HPV genomes in U2OS cells induces the ATR-dependent pathway, as shown by the accumulation of γH2AX, ATR-interacting protein (ATRIP), and topoisomerase IIβ-binding protein 1 (TopBP1) in viral replication centers. Viral oncogenes do not play a role in this activation, which is induced only through DNA replication or by replication proteins E1 and E2. The ATR pathway in viral replication centers is likely activated through DNA replication stress and might play an important role in engaging cellular DNA repair/recombination machinery for effective replication of the viral genome upon active amplification.     

Recombination-Dependent Oligomerization of Human Papillomavirus Genomes upon Transient DNA Replication

We describe the extensive and progressive oligomerization of human papillomavirus (HPV) genomes after transfection into the U2OS cell line. The HPV genomic oligomers are extrachromosomal concatemeric molecules containing the viral genome in a head-to-tail orientation. The process of oligomerization does not depend on the topology of the input DNA, and it does not require any other viral factors besides replication proteins E1 and E2. We provide evidence that oligomerization of the HPV18 and HPV11 genomes involves homologous recombination. We also demonstrate oligomerization of the HPV18 and HPV11 genomes in SiHa, HeLa, and C-33 A cell lines and provide examples of oligomeric HPV genomes in clinical samples obtained from HPV-infected patients.     

Concomitant epicardial left atrial appendage ligation and left atrial ablation of atrial fibrillation: Safety,feasibility and outcome

IntroductionWe present initial results of patients undergoing a combined procedure of epicardial LAA ligation in addition to left atrial ablation for AF.Methods9 patients were included for additional use of LARIAT as an individual treatment approach for AF. First an epicardial LAA ligation was performed, in the same procedure left atrial ablations consisting of PVI and additional substrate based modifying ablations were performed. Follow–up at 3 months and 12 months was performed.ResultsThere was only 1 minor procedural complication (11%) involving epicardial bleeding and 2 late adverse events of pericardial tamponade and stroke. At the final follow-up (median 20 months) 7 patients were in stable sinus rhythm (78%) and 2 pts had reduced AF burden.ConclusionConcomitant epicardial LAA ligation and ablation is feasible in selected patients with a reasonable risk profile. More prospective data are required to validate the safety and efficacy.     

Isolation and characterization of an apoptotic and platelet aggregation inhibiting L-amino acid oxidase from Vipera berus berus (common viper) venom

An L-amino acid oxidase was isolated from the venom of the common viper Vipera berus berus by a three-step procedure combining gel filtration, ion exchange and hydrophobic chromatography. The enzyme is a non-covalently bound homodimer with a monomeric molecular mass of 57.7 kDa. The N-terminal amino acid sequence and the internal peptide sequences show close structural homology with other snake venom L-amino acid oxidases. The purified protein catalyzed oxidative desamination of L-amino acids, the most specific substrate is L-Phe. The best substrates among the studied 20 amino acids were: L-Met, L-Leu, L-Phe, L-Ile, L-Arg and L-His. Five amino acids, L-Ser, L-Pro, Gly, L-Thr and L-Cys, were not oxidized. The enzyme inhibited ADP-induced platelet aggregation dose-dependently with an IC50 of 0.07 microM. The effect was neutralized by catalase. V. berus berus LAAO induced apoptosis in cultured HeLa and K562 cells as shown by DNA fragmentation gel pattern. The induction of apoptosis was inhibited by catalase.     

A trinuclear copper(II) complex with 2,5-pyridine-dicarboxylato bridges - [Cu3(2,5-pydc)2(Me5dien)2(H2O)2(BF4)2] · H2O: Synthesis, crystal structure and magnetic properties

A trinuclear copper(II) complex, [Cu₃(2,5-pydc)₂(Me₅dien)₂(BF₄)₂(H₂O)₂] · H₂O 1, has been constructed from 2,5-pyridine-dicarboxylato bridges (2,5-pydc²⁻) and N,N,N′,N″,N″-pentamethyl-diethylenetriamine (Me₅dien) acting as a blocking ligand. The copper ions, within the centrosymmetric trinuclear cations, are connected by two 2,5-pydc²⁻ bridges, with an intramolecular Cu···Cu separation of 8.432 Å. The central copper ion exhibits an elongated octahedral geometry, with semicoordinated ions, while the terminal ones are pentacoordinated (distorted square-pyramidal geometry). The cryomagnetic investigation of 1 reveals an antiferromagnetic coupling of the copper(II) ions (J = −5.9 cm⁻¹, H = −JS_Cu1S_Cu2 − JS_Cu2S_Cu1a).     

An evaluation of the performance of tag SNPs derived from HapMap in a Caucasian population

The Haplotype Map (HapMap) project recently generated genotype data for more than 1 million single-nucleotide polymorphisms (SNPs) in four population samples. The main application of the data is in the selection of tag single-nucleotide polymorphisms (tSNPs) to use in association studies. The usefulness of this selection process needs to be verified in populations outside those used for the HapMap project. In addition, it is not known how well the data represent the general population, as only 90–120 chromosomes were used for each population and since the genotyped SNPs were selected so as to have high frequencies. In this study, we analyzed more than 1,000 individuals from Estonia. The population of this northern European country has been influenced by many different waves of migrations from Europe and Russia. We genotyped 1,536 randomly selected SNPs from two 500-kbp ENCODE regions on Chromosome 2. We observed that the tSNPs selected from the CEPH (Centre d'Etude du Polymorphisme Humain) from Utah (CEU) HapMap samples (derived from US residents with northern and western European ancestry) captured most of the variation in the Estonia sample. (Between 90% and 95% of the SNPs with a minor allele frequency of more than 5% have an r² of at least 0.8 with one of the CEU tSNPs.) Using the reverse approach, tags selected from the Estonia sample could almost equally well describe the CEU sample. Finally, we observed that the sample size, the allelic frequency, and the SNP density in the dataset used to select the tags each have important effects on the tagging performance. Overall, our study supports the use of HapMap data in other Caucasian populations, but the SNP density and the bias towards high-frequency SNPs have to be taken into account when designing association studies.