Optimizing a PCR protocol for cpn60-based microbiome profiling of samples variously contaminated with host genomic DNA.

The current recommended protocol for chaperonin-60 (cpn60) universal target based microbiome profiling includes universal PCR of microbiome samples across an annealing temperature gradient to maximize the diversity of sequences amplified. However, the value of including this gradient approach has not been formally evaluated since the optimization of a modified universal PCR primer cocktail for cpn60 PCR. PCR conditions that maximize representation of the microbiome while minimizing PCR-associated distortion of the community structure, especially in samples containing large amounts of host genomic DNA are critical. The goal of this study was to measure the effects of PCR annealing temperature and the ratio of host to bacterial DNA on the outcome of microbiota analysis, using pig microbiota as a model environment.Six samples were chosen with an anticipated range of ratios of pig to bacterial genomic DNA, and universal cpn60 PCR amplification with an annealing temperature gradient was used to create libraries for pyrosequencing, resulting in 426,477 sequences from the six samples. The sequences obtained were classified as target (cpn60) or non-target based on the percent identity of their closest match to the cpnDB reference database, and target sequences were further processed to create microbiome profiles for each sample at each annealing temperature. Annealing temperature affected the amount of PCR product generated, with more product generated at higher temperatures. Samples containing proportionally more host genomic DNA yielded more non-target reads, especially at lower annealing temperatures. However, microbiome composition for each sample across the annealing temperature gradient remained consistent at both the phylum and operational taxonomic unit levels. Although some microbial sequences were detected at only one annealing temperature, these sequences accounted for a minority of the total microbiome.These results indicate that PCR annealing temperature does have an affect on cpn60 based microbiome profiles, but that most of the differences are due to differences in detection of low abundance sequences. Higher annealing temperatures resulted in larger amounts of PCR product and lower amounts of non-target sequence amplification, especially in samples containing proportionally large amounts of host DNA. Taken together these results provide important information to guide decisions about experimental design for cpn60 based microbiome studies

Approximation of Rough Functions

For given $p\in\lbrack1,\infty]$ and $g\in L^{p}\mathbb{(R)}$, we establish the existence and uniqueness of solutions $f\in L^{p}(\mathbb{R)}$, to the equation $$f(x)-af(bx)=g(x),$$ where $a\in\mathbb{R}$, $b\in\mathbb{R} \setminus \{0\}$, and $\left\vert a\right\vert \neq\left\vert b\right\vert ^{1/p}$. Solutions include well-known nowhere differentiable functions such as those of Bolzano, Weierstrass, Hardy, and many others. Connections and consequences in the theory of fractal interpolation, approximation theory, and Fourier analysis are established.Comment: 16 pages, 3 figure

Multibeam Laser Altimeter for Planetary Topographic Mapping

Laser altimetry provides an active, high-resolution, high-accuracy method for measurement of planetary and asteroid surface topography. The basis of the measurement is the timing of the roundtrip propagation of short-duration pulses of laser radiation between a spacecraft and the surface. Vertical, or elevation, resolution of the altimetry measurement is determined primarily by laser pulse width, surface-induced spreading in time of the reflected pulse, and the timing precision of the altimeter electronics. With conventional gain-switched pulses from solid-state lasers and nanosecond resolution timing electronics, submeter vertical range resolution is possible anywhere from orbital altitudes of approximately 1 km to altitudes of several hundred kilometers. Horizontal resolution is a function of laser beam footprint size at the surface and the spacing between successive laser pulses. Laser divergence angle and altimeter platform height above the surface determine the laser footprint size at the surface, while laser pulse repetition rate, laser transmitter beam configuration, and altimeter platform velocity determine the spacing between successive laser pulses. Multiple laser transmitters in a single laser altimeter instrument that is orbiting above a planetary or asteroid surface could provide across-track as well as along-track coverage that can be used to construct a range image (i.e., topographic map) of the surface. We are developing a pushbroom laser altimeter instrument concept that utilizes a linear array of laser transmitters to provide contiguous across-track and along-track data. The laser technology is based on the emerging monolithic combination of individual, 1-sq cm diode-pumped Nd:YAG laser pulse emitters. Details of the multi-emitter laser transmitter technology, the instrument configuration, and performance calculations for a realistic Discovery-class mission will be presented

High Energy Neutrinos and Photons from Curvature Pions in Magnetars

We discuss the relevance of the curvature radiation of pions in strongly magnetized pulsars or magnetars, and their implications for the production of TeV energy neutrinos detectable by cubic kilometer scale detectors, as well as high energy photons.Comment: 19 pages, 4 figures, to appear in JCA