EXPEDITED PCR WITH STIRRING

Disclosed are an apparatus and methods for rapid amplifi cation of nucleic acids . More particularly , the present dis closure relates to an apparatus for mixing a reaction solution during amplification of nucleic acids and to methods for amplifying nucleic acids . Also disclosed are methods for lysing cells in a sample and amplifying nucleic acids

A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide election

The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an impor-tant reaction of extraordinary complexity. The DNA polymerase does not merely facilitate the insertion of dNMP, but it also performs rapid screening of substrates to ensure a high degree of fidelity. Several kinetic studies have determined rate constants and equilibrium constants for the elementary steps that make up the overall pathway. The information is used to develop a macro-scopic kinetic model, using an approach described by Ninio [Ninio J., 1987. Alternative to the steady-state method: derivation of reaction rates from first-passage times and pathway probabili-ties. Proc. Natl. Acad. Sci. U.S.A. 84, 663–667]. The principle idea of the Ninio approach is to track a single template/primer complex over time and to identify the expected behavior. The average time to insert a single nucleotide is a weighted sum of several terms, in-cluding the actual time to insert a nucleotide plus delays due to polymerase detachment from ei-ther the ternary (template-primer-polymerase) or quaternary (+nucleotide) complexes and time delays associated with the identification and ultimate rejection of an incorrect nucleotide from the binding site. The passage times of all events and their probability of occurrence are ex-pressed in terms of the rate constants of the elementary steps of the reaction pathway. The model accounts for variations in the average insertion time with different nucleotides as well as the in-fluence of G+C content of the sequence in the vicinity of the insertion site. Furthermore the model provides estimates of error frequencies. If nucleotide extension is recognized as a compe-tition between successful insertions and time delaying events, it can be described as a binomial process with a probability distribution. The distribution gives the probability to extend a primer/template complex with a certain number of base pairs and in general it maps annealed complexes into extension products

Continuous low-intensity ultrasound attenuates IL-6 and TNFα-induced catabolic effects and repairs chondral fissures in bovine osteochondral explants

Background: Cartilage repair outcomes are compromised in a pro-inflammatory environment; therefore, the mitigation of pro-inflammatory responses is beneficial. Treatment with continuous low-intensity ultrasound (cLIUS) at the resonant frequency of 5 MHz is proposed for the repair of chondral fissures under pro-inflammatory conditions. Methods: Bovine osteochondral explants, concentrically incised to create chondral fissures, were maintained under cLIUS (14 kPa (5 MHz, 2.5 Vpp), 20 min, 4 times/day) for a period of 28 days in the presence or absence of cytokines, interleukin-6 (IL-6) or tumor necrosis factor (TNF)α. Outcome assessments included histological and immunohistochemical staining of the explants; and the expression of catabolic and anabolic genes by qRT-PCR in bovine chondrocytes. Cell migration was assessed by scratch assays, and by visualizing migrating cells into the hydrogel core of cartilage-hydrogel constructs. Results: Both in the presence and absence of cytokines, higher percent apposition along with closure of fissures were noted in cLIUS-stimulated explants as compared to non-cLIUS-stimulated explants on day 14. On day 28, the percent apposition was not significantly different between unstimulated and cLIUS-stimulated explants exposed to cytokines. As compared to non-cLIUS-stimulated controls, on day 28, cLIUS preserved the distribution of proteoglycans and collagen II in explants despite exposure to cytokines. cLIUS enhanced the cell migration irrespective of cytokine treatment. IL-6 or TNFα-induced increases in MMP13 and ADAMTS4 gene expression was rescued by cLIUS stimulation in chondrocytes. Under cLIUS, TNFα-induced increase in NF-κB expression was suppressed, and the expression of collagen II and TIMP1 genes were upregulated. Conclusion: cLIUS repaired chondral fissures, and elicited pro-anabolic and anti-catabolic effects, thus demonstrating the potential of cLIUS in improving cartilage repair outcomes

Solitary waves and supersonic reaction front in metastable solids

Motivated by an increasing number of remarkable experimental observations on the role of pressure and shear stress in solid reactions, explosions and detonations, we present a simple toy model that embodies nonlinear elasticity and dispersion as well as chemical or phase transformation. This generalization of the Toda Lattice provides an effective model for the description of the organization during an abrupt transformation in a solid. One of the challenges is to capture both the equilibrium degrees of freedom as well as to quantify the possible role of out-of-equilibrium perturbations. In the Toda Lattice, we verify that the particle velocities converge in distribution towards the Maxwell-Boltzmann distribution, thus allowing us to define a bona-fide temperature. In addition, the balance between nonlinearity and wave dispersion may create solitary waves that act as energy traps. In the presence of reactive chemistry, we show that the trapping of the released chemical energy in solitary waves that are excited by an initial perturbation provides a positive feedback that enhances the reaction rate and leads to supersonic explosion front propagation. These modes of rupture observed in our model may provide a first-order description of ultrafast reactions of heterogeneous mixtures under mechanical loading

Theoretically proposed optimal frequency for ultrasound induced cartilage restoration

Background: Matching the frequency of the driving force to that of the system’s natural frequency of vibration results in greater amplitude response. Thus we hypothesize that applying ultrasound at the chondrocyte’s resonant frequency will result in greater deformation than applying similar ultrasound power at a frequency outside of the resonant bandwidth. Based on this resonant hypothesis, our group previously confirmed theoretically and experimentally that ultrasound stimulation of suspended chondrocytes at resonance (5 MHz) maximized gene expression of load inducible genes. However, this study was based on suspended chondrocytes. The resonant frequency of a chondrocyte does not only depend on the cell mass and intracellular stiffness, but also on the mechanical properties of the surrounding medium. An in vivo chondrocyte’s environment differs whether it be a blood clot (following microfracture), a hydrogel or the pericellular and extracellular matrices of the natural cartilage. All have distinct structures and compositions leading to different resonant frequencies. In this study, we present two theoretical models, the first model to understand the effects of the resonant frequency on the cellular deformation and the second to identify the optimal frequency range for clinical applications of ultrasound to enhance cartilage restoration. Results: We showed that applying low-intensity ultrasound at the resonant frequency induced deformation equivalent to that experimentally calculated in previous studies at higher intensities and a 1 MHz frequency. Additionally, the resonant frequency of an in vivo chondrocyte in healthy conditions, osteoarthritic conditions, embedded in a blood clot and embedded in fibrin ranges from 3.5 − 4.8 MHz. Conclusion: The main finding of this study is the theoretically proposed optimal frequency for clinical applications of therapeutic ultrasound induced cartilage restoration is 3.5 − 4.8 MHz (the resonant frequencies of in vivo chondrocytes). Application of ultrasound in this frequency range will maximize desired bioeffects

Di-μ-hydroxido-bis­[tris­(4,4,4-trifluoro-1-phenyl­acetyl­acetonato-κ2 O,O′)hafnium(IV)] dimethyl­formamide disolvate

The binuclear molecule of the title compound, [Hf2(C10H6F3O2)6(OH)2]·2C3H7NO, lies across an inversion centre and contains a HfIV atom which is eight-coordinated and surrounded by three chelating β-diketonato tris­(4,4,4-trifluoro-1-phenyl­acetyl­acetonate (tfba−) ligands and two bridging OH− groups in a distorted square-anti­prismatic geometry. The Hf—O bond lengths vary from 2.073 (2) to 2.244 (2) Å and the O—Hf—O bite angles vary from 73.49 (9) to 75.60 (9)°. Weak O—H⋯O hydrogen-bonding inter­actions are observed between the bridging hy­droxy groups and the dimethylformamide solvent mol­ecules. The unit cell contains solvent-accessible voids of 131 Å3, but the residual electron density in the difference Fourier map suggests no solvent mol­ecule occupies this void

Tetra­kis(1,3-diphenyl­propane-1,3-dionato)hafnium(IV)

In the title compound, [Hf(C15H11O2)4], the HfIV atom is coordinated by four 1,3-diphenyl­propane-1,3-dionato ligands with an average Hf—O distance of 2.17 (3) Å and O—Hf—O bite angles varying from 74.5 (1) to 75.02 (9)°. The coordination polyhedron shows a slightly distorted Archimedean square-anti­prismatic geometry. The crystal packing is stabilized by weak C—H⋯O inter­actions

A Macrokinetic Study of the High-Temperature Solid-Phase Titanium-Carbon Reaction

An experimental method, electro thermal explosion (ETE), is used to measure the macro kinetic parameters of the high-temperature titanium/carbon reaction. Different stages of the reaction have been identified, but the focus of this study is on the reaction between solid titanium and solid carbon, i.e., prior to the melting of titanium. The reaction has high activation energy, and an electric current is used to heat the cylindrically shaped sample to a specified temperature. The current is shut off at a temperature below the melting point of titanium; any further temperature rise is only due to reaction. The output of the ETE equipment is temperature time data that can be processed to recover the kinetic parameters. The activation energy and preexponential factor of the reaction rate constant are calculated and comprise 214 kJ mol-1 and (6.2 ( 1.5) _ 107 s-1, respectively. An important aspect of solid-phase reactions is the contact area between reactants. The contact area between titanium and carbon particles is calculated, and the reaction constant is corrected for this effect

Tetra­kis(quinolin-8-olato-κ2 N,O)hafnium(IV) toluene disolvate

In the title compound, [Hf(C9H6NO)4]·2C7H8, the hafnium metal centre is coordinated by four N,O-donating bidentate quinolin-8-olate ligands arranged to give a square-anti­prismatic coordination polyhedron with a slightly distorted dodeca­hedral geometry. The average Hf—O and Hf—N distances are 2.096 (3) and 2.398 (3) Å, respectively, and the average O—Hf—N bite angle is 70.99 (11)°. The crystal packing is controlled by π–π inter­actions between quinoline ligands of neighbouring mol­ecules and hydrogen-bonding inter­actions. The inter­planar distances vary between 3.138 (1) and 3.208 (2) Å, while the centroid–centroid distances range from 3.576 (1) to 4.074 (1) Å

Mechanotransduction of Ultrasound is Frequency Dependent Below the Cavitation Threshold

This study provides evidence that low-intensity ultrasound directly affects nuclear processes, and the magnitude of the effect varies with frequency. In particular, we show that the transcriptional induction of first load-inducible genes, which is independent of new protein synthesis, is frequency dependent. Bovine chondrocytes were exposed to low-intensity below the cavitational threshold) ultrasound at 2,5 and 8 MHz. Ultrasound elevated the expression of early response genes c-Fos, c-Jun and c-Myc, maximized at 5 MHz. The phosphorylated ERK inhibitor PD98059 abrogated any increase in c-series gene expression, suggesting that signaling occurs via the MAPPK/ERK pathway. However, phosphorylated ERK levels did not change with ultrasound frequency, indicating that processes downstream of ERK phosphorylation (such as nuclear transport and chromatin reorganization) respond to ultrasound with frequency dependence. A quantitative, biphasic mathematical model based on Biot theory predicted that cytoplasmic and nuclear stress is maximized at 5.2 ± 0.8 MHz for a chondrocyte, confirming experimental measurements