The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters

Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest-neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching complementary sequence and other spots with one or two mismatches: in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays.Comment: 31 pages, 5 figure

Dynamics of Resonances in Strongly Interacting Systems

The effects of the propagation of particles which have a finite life-time and an according broad distribution in their mass spectrum are discussed in the context of a transport descriptions. In the first part some example cases of mesonic modes in nuclear matter at finite densities and temperatures are presented. These equilibrium calculations illustrate the dynamical range of spectral distributions to be adequately covered by non-equilibrium description of the dynamics of two nuclei colliding at high energies. The second part addresses the problem of transport descriptions which properly account for the damping width of the particles. A systematic and general gradient approximation is presented in the form of diagrammatic rules which permit to derive a self-consistent transport scheme from the Kadanoff--Baym equation. The scheme is conserving and thermodynamically consistent provided the self-energies are obtained within the Phi-derivable two-particle irreducible (2PI) method of Baym. The merits, the limitations and partial cures of the limitations of this transport scheme are discussed in detail.Comment: To appear in the proceedings of the International Conference "Progress in Nonequilibrium Green's Functions III", Kiel, 22.-26. August 200

Prognostic implications of left ventricular global longitudinal strain in heart failure patients with narrow QRS complex treated with cardiac resynchronization therapy: a subanalysis of the randomized EchoCRT trial

Aim: Left ventricular (LV) global longitudinal strain (GLS) reflects LV systolic function and correlates inversely with the extent of LV myocardial scar and fibrosis. The present subanalysis of the Echocardiography Guided CRT trial investigated the prognostic value of LV GLS in patients with narrow QRS complex. Methods and results: Left ventricular (LV) global longitudinal strain (GLS) was measured on the apical 2-, 4- and 3-chamber views using speckle tracking analysis. Measurement of baseline LV GLS was feasible in 755 patients (374 with cardiac resynchronization therapy (CRT)-ON and 381 with CRT-OFF). The median value of LV GLS in the overall population was 7.9%, interquartile range 6.2–10.1%. After a mean follow-up period of 19.4 months, 95 patients in the CRT-OFF group and 111 in the CRT-ON group reached the combined primary endpoint of all-cause mortality and heart failure hospitalization. Each 1% absolute unit decrease in LV GLS was independently associated with 11% increase in the risk to reach the primary endpoint (Hazard ratio 1.11; 95% confidence interval 95% 1.04–1.17, P < 0.001), after adjusting for ischaemic cardiomyopathy and randomization treatment among other clinically relevant variables. When categorizing patients according to quartiles of LV GLS, the primary endpoint occurred more frequently in patients in the lowest quartile (<6.2%) treated with CRT-ON vs. CRT-OFF (45.6% vs. 28.7%, P = 0.009) whereas, no differences were observed in patients with LV GLS ≥6.2% treated with CRT-OFF vs. CRT-ON (23.7% vs. 24.5%, respectively; P  = 0.62). Conclusion: Low LV GLS is associated with poor outcome in heart failure patients with QRS width <130 ms, independent of randomization to CRT or not. Importantly, in the group of patients with the lowest LV GLS quartile, CRT may have a detrimental effect on clinical outcomes

Acyclic Identification of Aptamers for Human alpha-Thrombin Using Over-Represented Libraries and Deep Sequencing

BACKGROUND: Aptamers are oligonucleotides that bind proteins and other targets with high affinity and selectivity. Twenty years ago elements of natural selection were adapted to in vitro selection in order to distinguish aptamers among randomized sequence libraries. The primary bottleneck in traditional aptamer discovery is multiple cycles of in vitro evolution. METHODOLOGY/PRINCIPAL FINDINGS: We show that over-representation of sequences in aptamer libraries and deep sequencing enables acyclic identification of aptamers. We demonstrated this by isolating a known family of aptamers for human α-thrombin. Aptamers were found within a library containing an average of 56,000 copies of each possible randomized 15mer segment. The high affinity sequences were counted many times above the background in 2-6 million reads. Clustering analysis of sequences with more than 10 counts distinguished two sequence motifs with candidates at high abundance. Motif I contained the previously observed consensus 15mer, Thb1 (46,000 counts), and related variants with mostly G/T substitutions; secondary analysis showed that affinity for thrombin correlated with abundance (K(d) = 12 nM for Thb1). The signal-to-noise ratio for this experiment was roughly 10,000∶1 for Thb1. Motif II was unrelated to Thb1 with the leading candidate (29,000 counts) being a novel aptamer against hexose sugars in the storage and elution buffers for Concanavilin A (K(d) = 0.5 µM for α-methyl-mannoside); ConA was used to immobilize α-thrombin. CONCLUSIONS/SIGNIFICANCE: Over-representation together with deep sequencing can dramatically shorten the discovery process, distinguish aptamers having a wide range of affinity for the target, allow an exhaustive search of the sequence space within a simplified library, reduce the quantity of the target required, eliminate cycling artifacts, and should allow multiplexing of sequencing experiments and targets

Quantum dot labeling of mesenchymal stem cells

<p>Abstract</p> <p>Background</p> <p>Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into bone, cartilage, fat and muscle cells and are being investigated for their utility in cell-based transplantation therapy. Yet, adequate methods to track transplanted MSCs <it>in vivo </it>are limited, precluding functional studies. Quantum Dots (QDs) offer an alternative to organic dyes and fluorescent proteins to label and track cells <it>in vitro </it>and <it>in vivo</it>. These nanoparticles are resistant to chemical and metabolic degradation, demonstrating long term photostability. Here, we investigate the cytotoxic effects of <it>in vitro </it>QD labeling on MSC proliferation and differentiation and use as a cell label in a cardiomyocyte co-culture.</p> <p>Results</p> <p>A dose-response to QDs in rat bone marrow MSCs was assessed in Control (no-QDs), Low concentration (LC, 5 nmol/L) and High concentration (HC, 20 nmol/L) groups. QD yield and retention, MSC survival, proinflammatory cytokines, proliferation and DNA damage were evaluated in MSCs, 24 -120 hrs post QD labeling. In addition, functional integration of QD labeled MSCs in an <it>in vitro </it>cardiomyocyte co-culture was assessed. A dose-dependent effect was measured with increased yield in HC vs. LC labeled MSCs (93 ± 3% vs. 50% ± 15%, p < 0.05), with a larger number of QD aggregates per cell in HC vs. LC MSCs at each time point (p < 0.05). At 24 hrs >90% of QD labeled cells were viable in all groups, however, at 120 hrs increased apoptosis was measured in HC vs. Control MSCs (7.2% ± 2.7% vs. 0.5% ± 0.4%, p < 0.05). MCP-1 and IL-6 levels doubled in HC MSCs when measured 24 hrs after QD labeling. No change in MSC proliferation or DNA damage was observed in QD labeled MSCs at 24, 72 and 120 hrs post labeling. Finally, in a cardiomyocyte co-culture QD labeled MSCs were easy to locate and formed functional cell-to-cell couplings, assessed by dye diffusion.</p> <p>Conclusion</p> <p>Fluorescent QDs label MSC effectively in an <it>in vitro </it>co-culture model. QDs are easy to use, show a high yield and survival rate with minimal cytotoxic effects. Dose-dependent effects suggest limiting MSC QD exposure.</p

Review of Speculative "Disaster Scenarios" at RHIC

We discuss speculative disaster scenarios inspired by hypothetical new fundamental processes that might occur in high energy relativistic heavy ion collisions. We estimate the parameters relevant to black hole production; we find that they are absurdly small. We show that other accelerator and (especially) cosmic ray environments have already provided far more auspicious opportunities for transition to a new vacuum state, so that existing observations provide stringent bounds. We discuss in most detail the possibility of producing a dangerous strangelet. We argue that four separate requirements are necessary for this to occur: existence of large stable strangelets, metastability of intermediate size strangelets, negative charge for strangelets along the stability line, and production of intermediate size strangelets in the heavy ion environment. We discuss both theoretical and experimental reasons why each of these appears unlikely; in particular, we know of no plausible suggestion for why the third or especially the fourth might be true. Given minimal physical assumptions the continued existence of the Moon, in the form we know it, despite billions of years of cosmic ray exposure, provides powerful empirical evidence against the possibility of dangerous strangelet production.Comment: 28 pages, REVTeX; minor revisions for publication (Reviews of Modern Physics, ca. Oct. 2000); email to [email protected]

Strange quark matter in a chiral SU(3) quark mean field model

We apply the chiral SU(3) quark mean field model to investigate strange quark matter. The stability of strange quark matter with different strangeness fraction is studied. The interaction between quarks and vector mesons destabilizes the strange quark matter. If the strength of the vector coupling is the same as in hadronic matter, strangelets can not be formed. For the case of beta equilibrium, there is no strange quark matter which can be stable against hadron emission even without vector meson interactions.Comment: 19 pages, 8 figure

Thermal dileptons at SPS energies

Clear signs of excess dileptons above the known sources were found at the SPS since long. However, a real clarification of these observations was only recently achieved by NA60, measuring dimuons with unprecedented precision in 158A GeV, In-In collisions. The excess mass spectrum in the region M<1 GeV is consistent with a dominant contribution from pi+pi- -> rho -> mu+mu- annihilation. The associated rho spectral function shows a strong broadening, but essentially no shift in mass. In the region M>1 GeV, the excess is found to be prompt, not due to enhanced charm production. The inverse slope parameter Teff associated with the transverse momentum spectra rises with mass up to the rho, followed by a sudden decline above. While the initial rise, coupled to a hierarchy in hadron freeze-out, points to radial flow of a hadronic decay source, the decline above signals a transition to a low-flow source, presumably of partonic origin. The mass spectra show at low transverse momenta the steep rise towards low masses characteristic for Planck-like radiation. The polarization of the excess referred to the Collins Soper frame is found to be isotropic. All observations are consistent with the interpretation of the excess as thermal radiation.Comment: Prepared for 20th International Conference on Ultra-Relativistic Nucleus-Nucleus Collisions: Quark Matter 2008 (QM2008), Jaipur, India, 4-10 Feb. 200