Phylogeny and Classification of the Trapdoor Spider Genus Myrmekiaphila: An Integrative Approach to Evaluating Taxonomic Hypotheses

Background

Revised by Bond and Platnick in 2007, the trapdoor spider genus Myrmekiaphila comprises 11 species. Species delimitation and placement within one of three species groups was based on modifications of the male copulatory device. Because a phylogeny of the group was not available these species groups might not represent monophyletic lineages; species definitions likewise were untested hypotheses. The purpose of this study is to reconstruct the phylogeny of Myrmekiaphila species using molecular data to formally test the delimitation of species and species-groups. We seek to refine a set of established systematic hypotheses by integrating across molecular and morphological data sets.

Methods and Findings

Phylogenetic analyses comprising Bayesian searches were conducted for a mtDNA matrix composed of contiguous 12S rRNA, tRNA-val, and 16S rRNA genes and a nuclear DNA matrix comprising the glutamyl and prolyl tRNA synthetase gene each consisting of 1348 and 481 bp, respectively. Separate analyses of the mitochondrial and nuclear genome data and a concatenated data set yield M. torreya and M. millerae paraphyletic with respect to M. coreyi and M. howelli and polyphyletic fluviatilis and foliata species groups.

Conclusions

Despite the perception that molecular data present a solution to a crisis in taxonomy, studies like this demonstrate the efficacy of an approach that considers data from multiple sources. A DNA barcoding approach during the species discovery process would fail to recognize at least two species (M. coreyi and M. howelli) whereas a combined approach more accurately assesses species diversity and illuminates speciation pattern and process. Concomitantly these data also demonstrate that morphological characters likewise fail in their ability to recover monophyletic species groups and result in an unnatural classification. Optimizations of these characters demonstrate a pattern of “Dollo evolution” wherein a complex character evolves only once but is lost multiple times throughout the group''s history.     

Rapid Transepithelial Transport of Prions following Inhalation

Prion infection and pathogenesis are dependent on the agent crossing an epithelial barrier to gain access to the recipient nervous system. Several routes of infection have been identified, but the mechanism(s) and timing of in vivo prion transport across an epithelium have not been determined. The hamster model of nasal cavity infection was used to determine the temporal and spatial parameters of prion-infected brain homogenate uptake following inhalation and to test the hypothesis that prions cross the nasal mucosa via M cells. A small drop of infected or uninfected brain homogenate was placed below each nostril, where it was immediately inhaled into the nasal cavity. Regularly spaced tissue sections through the entire extent of the nasal cavity were processed immunohistochemically to identify brain homogenate and the disease-associated isoform of the prion protein (PrP^d). Infected or uninfected brain homogenate was identified adhering to M cells, passing between cells of the nasal mucosa, and within lymphatic vessels of the nasal cavity at all time points examined. PrP^d was identified within a limited number of M cells 15 to 180 min following inoculation, but not in the adjacent nasal mucosa-associated lymphoid tissue (NALT). While these results support M cell transport of prions, larger amounts of infected brain homogenate were transported paracellularly across the respiratory, olfactory, and follicle-associated epithelia of the nasal cavity. These results indicate that prions can immediately cross the nasal mucosa via multiple routes and quickly enter lymphatics, where they can spread systemically via lymph draining the nasal cavity.     

Real-time Investigation of SV40 Large T-antigen Helicase Activity Using Surface Plasmon Resonance

The simian virus 40 (SV40) genome is a model system frequently employed for investigating eukaryotic replication. Large T-antigen (T-ag) is a viral protein responsible for unwinding the SV40 genome and recruiting necessary host factors prior to replication. In addition to duplex unwinding T-ag possesses G-quadruplex DNA helicase activity, the physiological consequence of which is unclear. However, formation of G-quadruplex DNA structures may be involved in genome maintenance and function, and helicase activity to resolve these structures may be necessary for efficient replication. We report the first real-time investigation of SV40 T-ag helicase activity using surface plasmon resonance (SPR). In the presence of ATP, T-ag was observed to bind to immobilized single-stranded DNA, forked duplex DNA, and the human telomeric foldover quadruplex DNA sequence. Inhibition of T-ag duplex helicase activity was observable in real-time and the intramolecular quadruplex was unwound.

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution,new validation tools,and implications for the future

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) “Resolution Revolution” made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.     

High‐throughput cloning and expression library creation for functional proteomics

The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single‐gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high‐throughput proteomics platforms, such as protein microarrays and cell‐based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high‐throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and Creator^TM DNA Cloning System) and compare them side‐by‐side. We also report an example of high‐throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12).     

Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions

The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.     

Vegetation and climate controls on potential CO2, DOC and DON production in northern latitude soils

Climatic change may influence decomposition dynamics in arctic and boreal ecosystems, affecting both atmospheric CO₂ levels, and the flux of dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) to aquatic systems. In this study, we investigated landscape‐scale controls on potential production of these compounds using a one‐year laboratory incubation at two temperatures (10° and 30 °C). We measured the release of CO₂, DOC and DON from tundra soils collected from a variety of vegetation types and climatic regimes: tussock tundra at four sites along a latitudinal gradient from the interior to the north slope of Alaska, and soils from additional vegetation types at two of those sites (upland spruce at Fairbanks, and wet sedge and shrub tundra at Toolik Lake in northern Alaska). Vegetation type strongly influenced carbon fluxes. The highest CO₂ and DOC release at the high incubation temperature occurred in the soils of shrub tundra communities. Tussock tundra soils exhibited the next highest DOC fluxes followed by spruce and wet sedge tundra soils, respectively. Of the fluxes, CO₂ showed the greatest sensitivity to incubation temperatures and vegetation type, followed by DOC. DON fluxes were less variable. Total CO₂ and total DOC release were positively correlated, with DOC fluxes approximately 10% of total CO₂ fluxes. The ratio of CO₂ production to DOC release varied significantly across vegetation types with Tussock soils producing an average of four times as much CO₂ per unit DOC released compared to Spruce soils from the Fairbanks site. Sites in this study released 80–370 mg CO₂‐C g soil C^?1 and 5–46 mg DOC g soil C^?1 at high temperatures. The magnitude of these fluxes indicates that arctic carbon pools contain a large proportion of labile carbon that could be easily decomposed given optimal conditions. The size of this labile pool ranged between 9 and 41% of soil carbon on a g soil C basis, with most variation related to vegetation type rather than climate.