Ex vivo biomechanical evaluation of polyester and polyblend suture techniques to perform equine laryngoplasty

Objective: To validate the use of a polyblend tape suture in equine laryngoplasty (PL). Study design: Experimental study. Animals: Thirty-two cadaveric larynges. Methods: Each larynx was randomly assigned to 1 of 4 groups: PL with polyblend tape suture (TigerTape), without (TT) or with a cannula (TTC) in the muscular process of the arytenoid cartilage, and PL with polyester suture (Ethibond), without (EB) or with a cannula (EBC). Construct stiffness, total migration, creep, and drift values were measured after 3000 cycles. The specimens were then loaded to failure to assess their residual properties: load at failure, total energy, displacement, and 2 stiffness coefficients. Results: After cyclic testing, the total migration and creep were lower in TTC (6.36 ± 1.20 mm; 1.35 ± 0.38 mm/s) than in EB (11.12 ± 4.20 mm; 3.39 ± 2.68 mm/s) and in the TT constructs (11.26 ± 1.49 mm; 3.20 ± 0.54 mm/s); however, no difference was found with EBC (9.19 ± 3.18 mm; 2.14 ± 0.99). A correlation was found between total migration and creep (R =.85). The TTC constructs failed at higher loads (129.51 ± 33.84 N) than EB (93.16 ± 18.21 N) and EBC (81.72 ± 13.26 N) whereas the EB and EBC constructs were less stiff than TT and TTC (P <.001). Conclusion: Biomechanical properties were generally superior for the TTC constructs tested under cyclical loading. The TT and TTC constructs failed at a higher load than EB and EBC constructs. The cannula in TTC and EBC reduced the failure at the muscular process. Clinical significance: These results provide evidence to support the in vivo evaluation of the polyblend tape suture with or without a cannula in the muscular process for laryngoplasty in horses

DNA unzipped under a constant force exhibits multiple metastable intermediates

Single molecule studies, at constant force, of the separation of double-stranded DNA into two separated single strands may provide information relevant to the dynamics of DNA replication. At constant applied force, theory predicts that the unzipped length as a function of time is characterized by jumps during which the strands separate rapidly, followed by long pauses where the number of separated base pairs remains constant. Here, we report previously uncharacterized observations of this striking behavior carried out on a number of identical single molecules simultaneously. When several single lphage molecules are subject to the same applied force, the pause positions are reproducible in each. This reproducibility shows that the positions and durations of the pauses in unzipping provide a sequence-dependent molecular fingerprint. For small forces, the DNA remains in a partially unzipped state for at least several hours. For larger forces, the separation is still characterized by jumps and pauses, but the double-stranded DNA will completely unzip in less than 30 min

Nonequilibrium effects in DNA microarrays: a multiplatform study

It has recently been shown that in some DNA microarrays the time needed to reach thermal equilibrium may largely exceed the typical experimental time, which is about 15h in standard protocols (Hooyberghs et al. Phys. Rev. E 81, 012901 (2010)). In this paper we discuss how this breakdown of thermodynamic equilibrium could be detected in microarray experiments without resorting to real time hybridization data, which are difficult to implement in standard experimental conditions. The method is based on the analysis of the distribution of fluorescence intensities I from different spots for probes carrying base mismatches. In thermal equilibrium and at sufficiently low concentrations, log I is expected to be linearly related to the hybridization free energy $\Delta G$ with a slope equal to $1/RT_{exp}$, where $T_{exp}$ is the experimental temperature and R is the gas constant. The breakdown of equilibrium results in the deviation from this law. A model for hybridization kinetics explaining the observed experimental behavior is discussed, the so-called 3-state model. It predicts that deviations from equilibrium yield a proportionality of $\log I$ to $\Delta G/RT_{eff}$. Here, $T_{eff}$ is an effective temperature, higher than the experimental one. This behavior is indeed observed in some experiments on Agilent arrays. We analyze experimental data from two other microarray platforms and discuss, on the basis of the results, the attainment of equilibrium in these cases. Interestingly, the same 3-state model predicts a (dynamical) saturation of the signal at values below the expected one at equilibrium.Comment: 27 pages, 9 figures, 1 tabl

Sex differences in clinical presentation, severity and outcome of stroke: Results from a hospital-based registry

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Single-molecule derivation of salt dependent base-pair free energies in DNA

Accurate knowledge of the thermodynamic properties of nucleic acids is crucial to predicting their structure and stability. To date most measurements of base-pair free energies in DNA are obtained in thermal denaturation experiments, which depend on several assumptions. Here we report measurements of the DNA base-pair free energies based on a simplified system, the mechanical unzipping of single DNA molecules. By combining experimental data with a physical model and an optimization algorithm for analysis, we measure the 10 unique nearest-neighbor base-pair free energies with 0.1 kcal mol-1 precision over two orders of magnitude of monovalent salt concentration. We find an improved set of standard energy values compared with Unified Oligonucleotide energies and a unique set of 10 base-pair-specific salt-correction values. The latter are found to be strongest for AA/TT and weakest for CC/GG. Our new energy values and salt corrections improve predictions of DNA unzipping forces and are fully compatible with melting temperatures for oligos. The method should make it possible to obtain free energies, enthalpies and entropies in conditions not accessible by bulk methodologies.Comment: Main text: 27 pages, 4 figures, 2 tables. Supporting Information: 51 pages, 19 figures, 4 table

A nonlinear dynamic model of DNA with a sequence-dependent stacking term

No simple model exists that accurately describes the melting behavior and breathing dynamics of double-stranded DNA as a function of nucleotide sequence. This is especially true for homogenous and periodic DNA sequences, which exhibit large deviations in melting temperature from predictions made by additive thermodynamic contributions. Currently, no method exists for analysis of the DNA breathing dynamics of repeats and of highly G/C- or A/T-rich regions, even though such sequences are widespread in vertebrate genomes. Here, we extend the nonlinear Peyrard–Bishop–Dauxois (PBD) model of DNA to include a sequence-dependent stacking term, resulting in a model that can accurately describe the melting behavior of homogenous and periodic sequences. We collect melting data for several DNA oligos, and apply Monte Carlo simulations to establish force constants for the 10 dinucleotide steps (CG, CA, GC, AT, AG, AA, AC, TA, GG, TC). The experiments and numerical simulations confirm that the GG/CC dinucleotide stacking is remarkably unstable, compared with the stacking in GC/CG and CG/GC dinucleotide steps. The extended PBD model will facilitate thermodynamic and dynamic simulations of important genomic regions such as CpG islands and disease-related repeats

New approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of Förster resonance energy transfer probes in 5′-nuclease assays

The article describes a new technology for real-time polymerase chain reaction (PCR) detection of nucleic acids. Similar to Taqman, this new method, named Snake, utilizes the 5′-nuclease activity of Thermus aquaticus (Taq) DNA polymerase that cleaves dual-labeled Förster resonance energy transfer (FRET) probes and generates a fluorescent signal during PCR. However, the mechanism of the probe cleavage in Snake is different. In this assay, PCR amplicons fold into stem–loop secondary structures. Hybridization of FRET probes to one of these structures leads to the formation of optimal substrates for the 5′-nuclease activity of Taq. The stem–loop structures in the Snake amplicons are introduced by the unique design of one of the PCR primers, which carries a special 5′-flap sequence. It was found that at a certain length of these 5′-flap sequences the folded Snake amplicons have very little, if any, effect on PCR yield but benefit many aspects of the detection process, particularly the signal productivity. Unlike Taqman, the Snake system favors the use of short FRET probes with improved fluorescence background. The head-to-head comparison study of Snake and Taqman revealed that these two technologies have more differences than similarities with respect to their responses to changes in PCR protocol, e.g. the variations in primer concentration, annealing time, PCR asymmetry. The optimal PCR protocol for Snake has been identified. The technology’s real-time performance was compared to a number of conventional assays including Taqman, 3′-MGB-Taqman, Molecular Beacon and Scorpion primers. The test trial showed that Snake supersedes the conventional assays in the signal productivity and detection of sequence variations as small as single nucleotide polymorphisms. Due to the assay’s cost-effectiveness and simplicity of design, the technology is anticipated to quickly replace all known conventional methods currently used for real-time nucleic acid detection

Single Molecule Statistics and the Polynucleotide Unzipping Transition

We present an extensive theoretical investigation of the mechanical unzipping of double-stranded DNA under the influence of an applied force. In the limit of long polymers, there is a thermodynamic unzipping transition at a critical force value of order 10 pN, with different critical behavior for homopolymers and for random heteropolymers. We extend results on the disorder-averaged behavior of DNA's with random sequences to the more experimentally accessible problem of unzipping a single DNA molecule. As the applied force approaches the critical value, the double-stranded DNA unravels in a series of discrete, sequence-dependent steps that allow it to reach successively deeper energy minima. Plots of extension versus force thus take the striking form of a series of plateaus separated by sharp jumps. Similar qualitative features should reappear in micromanipulation experiments on proteins and on folded RNA molecules. Despite their unusual form, the extension versus force curves for single molecules still reveal remnants of the disorder-averaged critical behavior. Above the transition, the dynamics of the unzipping fork is related to that of a particle diffusing in a random force field; anomalous, disorder-dominated behavior is expected until the applied force exceeds the critical value for unzipping by roughly 5 pN.Comment: 40 pages, 18 figure

MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering

Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating translational gene knockouts and (iii) introducing other coding or non-coding mutations, including amino acid substitutions, insertions, deletions and point mutations. The tool automatically designs oligos based on desired genotypic or phenotypic changes defined by the user, which can be used for high efficiency recombineering and MAGE. MODEST is available for free and is open to all users at http://modest.biosustain.dtu.dk