Membrane and Cytoplasmic Proteins of Mycobacterium avium subspecies paratuberculosis that Bind to Novel Monoclonal Antibodies

Monoclonal antibodies against Mycobacterium avium subspecies paratuberculosis (Map) proteins are important tools in Johne’s disease research and diagnostics. Johne’s disease is a chronic inflammatory intestinal disease of cattle, sheep, and other ruminant animals. We have previously generated multiple sets of monoclonal antibodies (mAbs) in different studies; however, because many were generated and screened against a whole-cell extract of Map, the antigens that bind to these antibodies remained unknown. In this study, we used three different approaches to identify the corresponding Map antigens for 14 mAbs that could not be identified previously. In the first approach, a new Map-lambda phage expression library was screened to identify corresponding antigens for 11 mAbs. This approach revealed that mAbs 7C8, 9H3, 12E4, 3G5, and 11B8 all detect MAP_3404 encoding the biotin carboxylase subunit of acetyl-CoA carboxylase, while mAbs 7A6, 11F8, and 10C12 detect the GroEL2 chaperonin (MAP_3936), 6C9 detects electron transfer flavoprotein (MAP_3060c), and 14G11 detects MAP_3976, a lipoprotein anchoring transpeptidase. The epitopes to a selection of these mAbs were also defined. In a second approach, MAP_2698c bound monoclonal antibody (mAb) 14D4 as determined using protein arrays. When both of these approaches failed to identify the antigen for mAb 12C9, immunoprecipitation, mass spectrometry analysis, and codon optimization was used to identify the membrane protein, MAP_4145, as the reacting antigen. Characterized antibodies were used to quickly interrogate mycobacterial proteomic preps. We conclude by providing a complete catalog of available mAbs to Map proteins, along with their cognate antigens and epitopes, if known. These antibodies are now thoroughly characterized and more useful for research and diagnostic purposes

A preparative mass spectrometer to deposit intact large native protein complexes

Electrospray ion-beam deposition (ES-IBD) is a versatile tool to study the structure and reactivity of molecules from small metal clusters to large protein assemblies. It brings molecules gently into the gas phase, where they can be accurately manipulated and purified, followed by controlled deposition onto various substrates. In combination with imaging techniques, direct structural information on well-defined molecules can be obtained, which is essential to test and interpret results from indirect mass spectrometry techniques. To date, ion-beam deposition experiments are limited to a small number of custom instruments worldwide, and there are no commercial alternatives. Here we present a module that adds ion-beam deposition capabilities to a popular commercial MS platform (Thermo Scientific Q Exactive UHMR mass spectrometer). This combination significantly reduces the overhead associated with custom instruments, while benefiting from established high performance and reliability. We present current performance characteristics including beam intensity, landing-energy control, and deposition spot size for a broad range of molecules. In combination with atomic force microscopy (AFM) and transmission electron microscopy (TEM), we distinguish near-native from unfolded proteins and show retention of the native shape of protein assemblies after dehydration and deposition. Further, we use an enzymatic assay to quantify the activity of a noncovalent protein complex after deposition on a dry surface. Together, these results not only indicate a great potential of ES-IBD for applications in structural biology, but also outline the challenges that need to be solved for it to reach its full potential

Consumers’ evaluation of allocation policies for scarce health care services: Vested interest activation trumps spatial and temporal distance

The allocation of scarce health care service resources often requires trade-offs between individual and collective outcomes (e.g., when some individuals benefit more strongly from a given policy th

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The current assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n=14) containing 32,928 and 36,697 protein-coding genes, respectively. The Petunia lineage has experienced at least two rounds of paleohexaploidization, the older gamma hexaploidy event, which is shared with other Eudicots, and the more recent Solanaceae paleohexaploidy event that is shared with tomato and other Solanaceae species. Transcription factors that were targets of selection during the shift from bee- to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral color patterns and pollination systems. The high quality genome sequences will enhance the value of Petunia as a model system for basic and applied research on a variety of unique biological phenomena

Moving through a dappled world: the aesthetics of shade and shadow in place

© 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group. In addressing geography’s neglect of shade and shadow, this paper explores how the dynamic play of shadow and light constitutes an integral part of everyday affective and sensory attunement to place and guides pedestrian movement. First, we identify how particular shadows are shaped by distinctive kinds of solar radiance, material forms, human visual perception and cultural representations. We then consider the different cultural ways in which shade and shadow have been interpreted across space and time and identify diverse shadowy effects in different geographical contexts. Thereafter, we focus on particular key elements of central Melbourne’s shadow aesthetics, discuss how patterns of shade guide urban choreographies, and explore how architects have imaginatively manipulated shadow

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

An Assessment of Sub-Meter Scale Spatial Variability of Arcellinida (Testate Lobose Amoebae) Assemblages in a Temperate Lake: Implications for Limnological Studies

Arcellinida (testate lobose amoebae), a group of benthic protists, were examined from 46 sediment-water interface samples collected from oligotrophic Oromocto Lake, New Brunswick, Canada. To assess (1) assemblage homogeneity at a sub-meter spatial scale and (2) the necessity for collecting samples from multiple stations during intra-lake surveys; multiple samples were collected from three stations (quadrats 1, 2, and 3) across the north basin of Oromocto Lake, with quadrat 1 (n = 16) being the furthest to the west, quadrat 2 (n = 15) situated closer to the center of the basin, and quadrat 3 (n = 15) positioned 300 m south of the mouth of Dead Brook, an inlet stream. Results from cluster analysis and non-metric multidimensional scaling (NMDS) analysis identified two major Arcellinida assemblages, A1 and A2, the latter containing two sub-assemblages (A2a and A2b). Redundancy analysis and variance partitioning results indicated that seven statistically significant environmental variables (K, S, Sb, Ti, Zn, Fe, and Mn) explained 41.5% of the total variation in the Arcellinida distribution. Iron, Ti and K, indicators of detrital runoff, had the greatest influence on assemblage variance. The results of this study reveal that closely spaced samples (~ 10 cm) in an open-water setting are comprised of homogenous arcellinidan assemblages, indicating that replicate sampling is not required. The results, however, must be tempered with respect to the various water properties and physical characteristics that comprise individual lakes as collection of several samples may likely be necessary when samplin

Sequential sample reservoirs for Itrax-XRF analysis of discrete samples

Geochemical analysis of sediment samples can be used to characterize between- and within-lake variability and provide insights into lake chemistry, depositional processes and contamination sources. The number of samples for geochemical studies is restricted by cost, sample volume required, and the destructive nature of inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, or wavelength dispersive x-ray fluorescence. Core scanners that incorporate energy dispersive x-ray fluorescence spectrometry, such as the Cox Itrax-XRF core scanner, have high through-put and can be used to produce high-quality geochemical datasets at low cost without destroying sample material. Here we describe a new analysis vessel that enables rapid, non-destructive Itrax-XRF analysis of discrete sediment samples