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.     

Recurrent Tissue-Specific mtDNA Mutations Are Common in Humans

Mitochondrial DNA (mtDNA) variation can affect phenotypic variation; therefore, knowing its distribution within and among individuals is of importance to understanding many human diseases. Intra-individual mtDNA variation (heteroplasmy) has been generally assumed to be random. We used massively parallel sequencing to assess heteroplasmy across ten tissues and demonstrate that in unrelated individuals there are tissue-specific, recurrent mutations. Certain tissues, notably kidney, liver and skeletal muscle, displayed the identical recurrent mutations that were undetectable in other tissues in the same individuals. Using RFLP analyses we validated one of the tissue-specific mutations in the two sequenced individuals and replicated the patterns in two additional individuals. These recurrent mutations all occur within or in very close proximity to sites that regulate mtDNA replication, strongly implying that these variations alter the replication dynamics of the mutated mtDNA genome. These recurrent variants are all independent of each other and do not occur in the mtDNA coding regions. The most parsimonious explanation of the data is that these frequently repeated mutations experience tissue-specific positive selection, probably through replication advantage.     

Evidence for a dinuclear active site in the metallo-beta-lactamase BcII with substoichiometric Co(II). A new model for metal uptake

Metallo-beta-lactamases are zinc-dependent enzymes that constitute one of the main resistance mechanisms to beta-lactam antibiotics. Metallo-beta-lactamases have been characterized both in mono- and dimetallic forms. Despite many studies, the role of each metal binding site in substrate binding and catalysis is still unclear. This is mostly due to the difficulties in assessing the metal content and site occupancy in solution. For this reason, Co(II) has been utilized as a useful probe of the active site structure. We have employed UV-visible, EPR, and NMR spectroscopy to study Co(II) binding to the metallo-beta-lactamase BcII from Bacillus cereus. The spectroscopic features were attributed to the two canonical metal binding sites, the 3H (His(116), His(118), and His(196)) and DCH (Asp(120), Cys(221), and His(263)) sites. These data clearly reveal the coexistence of mononuclear and dinuclear Co(II)-loaded forms at Co(II)/enzyme ratios as low as 0.6. This picture is consistent with the macroscopic dissociation constants here determined from competition binding experiments. A spectral feature previously assigned to the DCH site in the dinuclear species corresponds to a third, weakly bound Co(II) site. The present work emphasizes the importance of using different spectroscopic techniques to follow the metal content and localization during metallo-beta-lactamase turnover.     

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.     

Stand-level management of plantations to improve biodiversity values

As conservation reserves expand, the likelihood that they will capture areas degraded by previous land use increases. Ecological restoration of such areas will therefore play an increasing role in biodiversity conservation. On the New South Wales North Coast, recent expansion in the conservation estate has captured over 300 softwood and hardwood plantations, many with understoreys dominated by exotic weeds. Here we present an overview of the practices we have adopted in managing flooded gum (Eucalyptus grandis) plantations infested with lantana (Lantana camara) to enhance their biodiversity value. Experiments designed to overcome barriers limiting regeneration of native forest in conjunction with measurement of soil and plant responses yielded insights into the management of former timber plantations for biodiversity. Canonical Correspondence Analysis indicated that the level of canopy retention (or logging intensity) within sites consistently explained the greatest amount of variation in plant community composition (32–38% post-treatment). Thinning and burning stimulated regeneration of native species. Retained canopy cover was proportional to the richness or abundance of native woody shrubs, understorey trees and native perennial herbs, indicating that management intensity can be varied to promote a range of conservation values. A state-and-transition model summarising purported management actions and likely outcomes for these plantations is presented. This is the first time plantations have been managed solely for biodiversity. Logging income means that plantation restoration can be cost-neutral, and the positive influence of a cover crop of trees means that plantation management may generally be manipulated to promote biodiversity conservation.     

Horizontal transfer of a nitrate assimilation gene cluster and ecological transitions in fungi: a phylogenetic study

High affinity nitrate assimilation genes in fungi occur in a cluster (fHANT-AC) that can be coordinately regulated. The clustered genes include nrt2, which codes for a high affinity nitrate transporter; euknr, which codes for nitrate reductase; and NAD(P)H-nir, which codes for nitrite reductase. Homologs of genes in the fHANT-AC occur in other eukaryotes and prokaryotes, but they have only been found clustered in the oomycete Phytophthora (heterokonts). We performed independent and concatenated phylogenetic analyses of homologs of all three genes in the fHANT-AC. Phylogenetic analyses limited to fungal sequences suggest that the fHANT-AC has been transferred horizontally from a basidiomycete (mushrooms and smuts) to an ancestor of the ascomycetous mold Trichoderma reesei. Phylogenetic analyses of sequences from diverse eukaryotes and eubacteria, and cluster structure, are consistent with a hypothesis that the fHANT-AC was assembled in a lineage leading to the oomycetes and was subsequently transferred to the Dikarya (Ascomycota+Basidiomycota), which is a derived fungal clade that includes the vast majority of terrestrial fungi. We propose that the acquisition of high affinity nitrate assimilation contributed to the success of Dikarya on land by allowing exploitation of nitrate in aerobic soils, and the subsequent transfer of a complete assimilation cluster improved the fitness of T. reesei in a new niche. Horizontal transmission of this cluster of functionally integrated genes supports the "selfish operon" hypothesis for maintenance of gene clusters.