Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis

We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein- containing bands or spots are cut out and inserted directly into a gas- phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150- pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels

Indecision Trees: Learning Argument-Based Reasoning under Quantified Uncertainty

Using Machine Learning systems in the real world can often be problematic, with inexplicable black-box models, the assumed certainty of imperfect measurements, or providing a single classification instead of a probability distribution. This paper introduces Indecision Trees, a modification to Decision Trees which learn under uncertainty, can perform inference under uncertainty, provide a robust distribution over the possible labels, and can be disassembled into a set of logical arguments for use in other reasoning systems.Comment: 12 pages, 1 figur

Restoration and Reexamination of Apollo Lunar Dust Detector Data from Original Telemetry Files

We are recovering the original telemetry (Figure I) from the Apollo Dust, Thermal, Radiation Environment Monitor (DTREM) experiment, more commonly known as the Dust Detector, and producing full time resolution (54 second) data sets for release through the Planetary Data System (PDS). The primary objective of the experiment was to evaluate the effect of dust deposition, temperature, and radiation damage on solar cells on the lunar surface. The monitor was a small box consisting of three solar cells and thermistors mounted on the ALSEP (Apollo Lunar Surface Experiments Package) central station. The Dust Detector was carried on Apollo's 11, 12, 14 and 15. The Apollo 11 DTREM was powered by solar cells and only operated for a few months as planned. The Apollo 12, 14, and 15 detectors operated for 5 to 7 years, returning data every 54 seconds, consisting of voltage outputs from the three solar cells and temperatures measured by the three thermistors. The telemetry was received at ground stations and held on the Apollo Housekeeping (known as "Word 33") tapes. made available to the National Space Science Data Center (NSSDC) by Yosio Nakamura (University of Texas Institute for Geophysics). We have converted selected parts of the telemetry into uncalibrated and calibrated output voltages and temperatures

Targeting tauopathy with engineered tau-degrading intrabodies

BACKGROUND: The accumulation of pathological tau is the main component of neurofibrillary tangles and other tau aggregates in several neurodegenerative diseases, referred to as tauopathies. Recently, immunotherapeutic approaches targeting tau have been demonstrated to be beneficial in decreasing tauopathy in animal models. We previously found that passive immunotherapy with anti-tau antibody to human tau or expression of an anti-tau secreted single-chain variable fragment (scFv) in the central nervous system of a mouse model of tauopathy decreased but did not remove all tau-associated pathology. Although these and other studies demonstrate that conventional immunotherapeutic approaches targeting tau can influence tau pathogenesis, the majority of pathological tau remains in the cytosol of cells, not typically accessible to an extracellular antibody. Therefore, we reasoned targeting intracellular tau might be more efficacious in preventing or decreasing tauopathy. METHODS: By utilizing our anti-tau scFv, we generated anti-tau intrabodies for the expression in the cytosol of neurons. To enhance the degradation capacity of conventional intrabodies, we engineered chimeric anti-tau intrabodies fused to ubiquitin harboring distinct mutations that shuttle intracellular tau for either the proteasome or lysosomal mediated degradation. To evaluate the efficacy in delaying or eliminating tauopathy, we expressed our tau degrading intrabodies or controls in human tau transgenic mice by adeno-associated virus prior to overt tau pathology and after tau deposition. RESULTS: Our results demonstrate, the expression of chimeric anti-tau intrabodies significantly reduce tau protein levels in primary neuronal cultures expression human tau relative to a non-modified anti-tau intrabody. We found the expression of engineered tau-degrading intrabodies destined for proteasomal-mediated degradation are more effective in delaying or eliminating tauopathy than a conventional intrabody in aged human tau transgenic mice. CONCLUSION: This study, harnesses the strength of intrabodies that are amendable for targeting specific domains or modifications with the cell-intrinsic mechanisms that regulate protein degradation providing a new immunotherapeutic approach with potentially improved efficacy

The complementary niches of anthropocentric and biocentric conservationists

A divergence of values has become apparent in recent debates between conservationists who focus on ecosystem services that can improve human well-being and those who focus on avoiding the extinction of species. These divergent points of view fall along

Restoration and Reexamination of Data from the Apollo 11, 12, 14, and 15 Dust, Thermal and Radiation Engineering Measurements Experiments

As part of an effort by the Lunar Data Node (LDN) we are restoring data returned by the Apollo Dust, Thermal, and Radiation Engineering Measurements (DTREM) packages emplaced on the lunar surface by the crews of Apollo 11, 12, 14, and 15. Also commonly known as the Dust Detector experiments, the DTREM packages measured the outputs of exposed solar cells and thermistors over time. They operated on the surface for up to nearly 8 years, returning data every 54 seconds. The Apollo 11 DTREM was part of the Early Apollo Surface Experiments Package (EASEP), and operated for a few months as planned following emplacement in July 1969. The Apollo 12, 14, and 15 DTREMs were mounted on the central station as part of the Apollo Lunar Surface Experiments Package (ALSEP) and operated from deployment until ALSEP shutdown in September 1977. The objective of the DTREM experiments was to determine the effects of lunar and meteoric dust, thermal stresses, and radiation exposure on solar cells. The LDN, part of the Geosciences Node of the Planetary Data System (PDS), operates out of the National Space Science Data Center (NSSDC) at Goddard Space Flight Center. The goal of the LDN is to extract lunar data stored on older media and/or in obsolete formats, restore the data into a usable digital format, and archive the data with PDS and NSSDC. For the DTREM data we plan to recover the raw telemetry, translate the raw counts into appropriate output units, and then apply calibrations. The final archived data will include the raw, translated, and calibrated data and the associated conversion tables produced from the microfilm, as well as ancillary supporting data (metadata) packaged in PDS format

The Linear Theory Power Spectrum from the Lyman-alpha Forest in the Sloan Digital Sky Survey

We analyze the SDSS Ly-alpha forest P_F(k,z) measurement to determine the linear theory power spectrum. Our analysis is based on fully hydrodynamic simulations, extended using hydro-PM simulations. We account for the effect of absorbers with damping wings, which leads to an increase in the slope of the linear power spectrum. We break the degeneracy between the mean level of absorption and the linear power spectrum without significant use of external constraints. We infer linear theory power spectrum amplitude Delta^2_L(k_p=0.009s/km,z_p=3.0)=0.452_{-0.057-0.116}^{+0.069+0.141} and slope n_eff=-2.321_{-0.047-0.102}^{+0.055+0.131} (possible systematic errors are included through nuisance parameters in the fit - a factor >~5 smaller errors would be obtained on both parameters if we ignored modeling uncertainties). The errors are correlated and not perfectly Gaussian, so we provide a chi^2 table to accurately describe the results. The result corresponds to sigma_8=0.85, n=0.94, for a LCDM model with Omega_m=0.3, Omega_b=0.04, and h=0.7, but is most useful in a combined fit with the CMB. The inferred curvature of the linear power spectrum and the evolution of its amplitude and slope with redshift are consistent with expectations for LCDM models, with the evolution of the slope, in particular, being tightly constrained. We use this information to constrain systematic contamination, e.g., fluctuations in the UV background. This paper should serve as a starting point for more work to refine the analysis, including technical improvements such as increasing the size and number of the hydrodynamic simulations, and improvements in the treatment of the various forms of feedback from galaxies and quasars.Comment: Improved presentation, including fit results for (z). Simple code to produce LyaF chi^2 given linear power spectrum available at: http://www.cita.utoronto.ca/~pmcdonal/code.htm

The Lyman-alpha Forest Power Spectrum from the Sloan Digital Sky Survey

We measure the power spectrum, P_F(k,z), of the transmitted flux in the Ly-alpha forest using 3035 high redshift quasar spectra from the Sloan Digital Sky Survey. This sample is almost two orders of magnitude larger than any previously available data set, yielding statistical errors of ~0.6% and ~0.005 on, respectively, the overall amplitude and logarithmic slope of P_F(k,z). This unprecedented statistical power requires a correspondingly careful analysis of the data and of possible systematic contaminations in it. For this purpose we reanalyze the raw spectra to make use of information not preserved by the standard pipeline. We investigate the details of the noise in the data, resolution of the spectrograph, sky subtraction, quasar continuum, and metal absorption. We find that background sources such as metals contribute significantly to the total power and have to be subtracted properly. We also find clear evidence for SiIII correlations with the Ly-alpha forest and suggest a simple model to account for this contribution to the power. While it is likely that our newly developed analysis technique does not eliminate all systematic errors in the P_F(k,z) measurement below the level of the statistical errors, our tests indicate that any residual systematics in the analysis are unlikely to affect the inference of cosmological parameters from P_F(k,z). These results should provide an essential ingredient for all future attempts to constrain modeling of structure formation, cosmological parameters, and theories for the origin of primordial fluctuations.Comment: 92 pages, 45 of them figures, submitted to ApJ, data available at http://feynman.princeton.edu/~pmcdonal/LyaF/sdss.htm