Inexpensive Computation of the Inverse of the Genomic Relationship Matrix in Populations with Small Effective Population Size

Many computations with SNP data including genomic evaluation, parameter estimation, and genome-wide association studies use an inverse of the genomic relationship matrix. The cost of a regular inversion is cubic and is prohibitively expensive for large matrices. Recent studies in cattle demonstrated that the inverse can be computed in almost linear time by recursion on any subset of ∼10,000 individuals. The purpose of this study is to present a theory of why such a recursion works and its implication for other populations. Assume that, because of a small effective population size, the additive information in a genotyped population has a small dimensionality, even with a very large number of SNP markers. That dimensionality is visible as a limited number of effective SNP effects, independent chromosome segments, or the rank of the genomic relationship matrix. Decompose a population arbitrarily into core and noncore individuals, with the number of core individuals equal to that dimensionality. Then, breeding values of noncore individuals can be derived by recursions on breeding values of core individuals, with coefficients of the recursion computed from the genomic relationship matrix. A resulting algorithm for the inversion called “algorithm for proven and young” (APY) has a linear computing and memory cost for noncore animals. Noninfinitesimal genetic architecture can be accommodated through a trait-specific genomic relationship matrix, possibly derived from Bayesian regressions. For populations with small effective population size, the inverse of the genomic relationship matrix can be computed inexpensively for a very large number of genotyped individuals.     

Protonation of excited state pyrene-1-carboxylate by phosphate and organic acids in aqueous solution studied by fluorescence spectroscopy

Pyrene-1-carboxylic acid has a pK of 4.0 in the ground state and 8.1 in the singlet electronic excited state. In the pH range of physiological interest (pH approximately 5-8), the ground state compound is largely ionized as pyrene-1-carboxylate, but protonation of the excited state molecule occurs when a proton donor reacts with the carboxylate during the excited state lifetime of the fluorophore. Both forms of the pyrene derivatives are fluorescent, and in this work the protonation reaction was measured by monitoring steady-state and time-resolved fluorescence. The rate of protonation of pyrene-COO(-) by acetic, chloroacetic, lactic, and cacodylic acids is a function of DeltapK, as predicted by Marcus theory. The rate of proton transfer from these acids saturates at high concentration, as expected for the existence of an encounter complex. Trihydrogen-phosphate is a much better proton donor than dihydrogen- and monohydrogen-phosphate, as can be seen by the pH dependence. The proton-donating ability of phosphate does not saturate at high concentrations, but increases with increasing phosphate concentration. We suggest that enhanced rate of proton transfer at high phosphate concentrations may be due to the dual proton donating and accepting nature of phosphate, in analogy to the Grotthuss mechanism for proton transfer in water. It is suggested that in molecular structures containing multiple phosphates, such as membrane surfaces and DNA, proton transfer rates will be enhanced by this mechanism.     

Leukocyte trafficking and pain behavioral responses to a hydrogen sulfide donor in acute monoarthritis

Hydrogen sulfide (H(2)S) is an endogenous gaseous mediator with the ability to modulate tissue inflammation and pain. The aim of this study was to determine the effect of an H(2)S donor (Na(2)S) on leukocyte-endothelium interactions, blood flow, and pain sensation in acutely inflamed knee joints. Acute arthritis was induced in urethane anesthetized C57bl/6 mice by intra-articular injection of kaolin/carrageenan (24-h recovery), and the effect of local administration of Na(2)S on leukocyte trafficking was measured by intravital microscopy. Synovial blood flow was measured in inflamed knees by laser Doppler perfusion imaging. Finally, the effect of an intra-articular injection of Na(2)S on joint pain in control and inflamed rats was determined by hindlimb incapacitance and von Frey hair algesiometry. Local administration of an H(2)S donor to inflamed knees caused a dose-dependent reduction in leukocyte adherence and an increase in leukocyte velocity. These effects could be inhibited by coadministration of the ATP-sensitive K(+) channel blocker glibenclamide. Local administration of Na(2)S to inflamed joints caused a pronounced vasoconstrictor response; however, there was no observable effect of Na(2)S on joint pain. These findings establish H(2)S as a novel signaling molecule in rodent knee joints. H(2)S exhibits potent anti-inflammatory properties, but with no detectable effect on joint pain.     

CARDIOVASCULAR DISEASE: PREVENTION AND REHABILITATION

Cardiovascular related diseases are the major cause of death in the United States. The primary goal of the physician should be prevention of these diseases; however, once problems occur, a definitive rehabilitation program must be instituted in addition to the medical and surgical treatment. Although risk factors for the development of cardiovascular disease are numerous, statistical reports suggest that control of these factors might delay or prevent such development. This presentation describes the recommended Physical Medicine and Rehabilitation Program for high-risk patients or those with existing cardiovascular disease. The basis of the physical fitness and rehabilitation program is controlled progression-of-energy-expenditure physical activity and exercise in metabolic equivalents (METS). Education plays a key role in decreasing the incidence of cardiovascular disease, and should start at an early age. Adults at high risk of developing cardiovascular disease should be offered medically supervised physical fitness exercise programs, with monitoring where necessary.     

Silver particles enhance emission of fluorescent DNA oligomers

Here we describe a new opportunity in methodology for increasing the detectability of fluorescently labeled DNA on solid substrates. We show that the use of glass substrates coated with metallic silver particles results in an approximate 5-fold increase in the intensity of Cy3- or Cy5-labeled DNA oligomers. Proximity to these silver particles also increases the photostability of Cy3- and Cy5-labeled oligomers. These results suggest the use of DNA array substrates with silver particles for increased sensitivity in genetic analysis.     

Metal-enhanced fluorescence immunoassays using total internal reflection and silver island-coated surfaces

We present a generic immunoassay platform that uses enhanced total internal reflection fluorescence in the proximity of silver island films (SIFs), a surface coating consisting of metal (silver) particles. This platform is used with a model immunoassay where a protein antigen, rabbit immunoglobulin G, was immobilized on the SIF-coated glass surface. The signal from a fluorescent dye-labeled anti-rabbit antibody binding to the surface antigen was detected; different color dyes have been tested. Close placement of the fluorophore to surface-bound silver nanostructures results in dramatic signal enhancement (up to 40-fold) on the SIFs as compared with the glass slides. Use of the total internal reflection mode of excitation has significant advantages (over classic front-face excitation) for practical assay development. The limited evanescent wave excitation volume makes it possible to minimize the background signal and use the immunoassay with no need for any washing steps.     

Increased resonance energy transfer between fluorophores bound to DNA in proximity to metallic silver particles

We examined the effects of metallic silver particles on resonance energy transfer (RET) between fluorophores covalently bound to DNA. A coumarin donor and a Cy3 acceptor were positioned at opposite ends of a 23-bp double helical DNA oligomer. In the absence of silver particles the extent of RET is near 9%, consistent with a Forster distance R(0) near 50 A and a donor to acceptor distance near 75 A. The transfer efficiency increased when the solution of AMCA-DNA-Cy3 was placed between two quartz plates coated with silver island films to near 64%, as determined by both steady-state and time-resolved measurements. The apparent R(0) in the presence of silver island films increases to about 110 A. These values of the transfer efficiency and R(0) represent weighted averages for donor-acceptor pairs near and distant from the metallic surfaces, so that the values at an optimal distance are likely to be larger. The increased energy transfer is observed only between two sandwiched silvered slides. When we replaced one silvered slide with a quartz plate the effect vanished. Also, the increased energy transfer was not observed for silvered slides separated more than a few micrometers. These results suggest the use of metal-enhanced RET in PCR, hybridization, and other DNA assays, and the possibility of controlling energy transfer by the distance between silver surfaces.     

Directional surface plasmon-coupled emission: A new method for high sensitivity detection

Fluorescence emission is nearly isotropic in space. With typical optical components the collection efficiency is 1% or less. In this preliminary report, we describe a novel approach to transforming the normally isotropic emission into directional emission with a collection efficiency near 50%. This can be accomplished for fluorophores located near a semi-transparent silver film on a glass substrate. The emission couples with the surface plasmon resonance on the silver surface and enters the transparent substrate at a sharply defined angle, the surface plasmon angle for the emission wavelength. We estimate that 40-70% of the total emission enters the substrate at the plasmon angle and can thus be directed towards a detector. Background emission from fluorophores distant from the silver does not couple with the plasmon and is not detected. Different emission wavelengths couple at different angles allowing spectral discrimination without additional optics. Surface plasmon-coupled emission represents a new technology which can be used for high detection efficiency with microfluidic and/or surface-bound assay formats.     

DNA hybridization assays using metal-enhanced fluorescence

We describe a new approach to DNA hybridization assays using metal-enhanced fluorescence. Thiolated oligonucleotides were bound to silver particles on a glass substrate. Addition of a complementary fluorescein-labeled oligonucleotide resulted in a dramatic time-dependent 12-fold increase in fluorescence intensity during hybridization. Proximity to silver particles resulted in a decreased fluorescence lifetime. This effect is thought to be the result of enhanced fluorescence from fluorescein near metallic silver particles. Hybridization could thus be measured from the decay kinetics of the emission, which can be measured independently from the emission intensity. These results suggest the use of silver particles as a general approach to measure DNA hybridization as a method to increase the sensitivity of DNA detection.