Sampling accuracy of reef resource inventory technique

A new technique called the reef resource inventory (RRI) was developed to map the distribution and abundance of benthos and substratum on reefs. The rapid field sampling technique uses divers to visually estimate the percentage cover of categories of benthos and substratum along 2×20 m plotless strip-transects positioned randomly over the tops, and systematically along the edge of reefs. The purpose of this study was to compare the relative sampling accuracy of the RRI against the line intercept transect technique (LIT), an international standard for sampling reef benthos and substratum. Analysis of paired sampling with LIT and RRI at 51 sites indicated sampling accuracy was not different (P>0.05) for 8 of the 12 benthos and substratum categories used in the study. Significant differences were attributed to small-scale patchiness and cryptic coloration of some benthos; effects associated with sampling a sparsely distributed animal along a line versus an area; difficulties in discriminating some of the benthos and substratum categories; and differences due to visual acuity since LIT measurements were taken by divers close to the seabed whereas RRI measurements were taken by divers higher in the water column. The relative cost efficiency of the RRI technique was at least three times that of LIT for all benthos and substratum categories and as much as 10 times higher for two categories. These results suggest that the RRI can be used to obtain reliable and accurate estimates of relative abundance of broad categories of reef benthos and substratum.

Regulation of Mycobacterium-specific mononuclear cell responses by 25-hydroxyvitamin D3

The active vitamin D metabolite, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to be an important regulator of innate and adaptive immune function. In addition, synthesis of 1,25(OH)(2)D(3) from 25-hydroxyvitamin D(3) (25(OH)D(3)) by the enzyme 1α-hydroxylase in monocytes upon activation by TLR signaling has been found to regulate innate immune responses of monocytes in an intracrine fashion. In this study we wanted to determine what cells expressed 1α-hydroxylase in stimulated peripheral blood mononuclear cell (PBMC) cultures and if conversion of 25(OH)D(3) to 1,25(OH)(2)D(3) in PBMC cultures regulated antigen-specific immune responses. Initially, we found that stimulation of PBMCs from animals vaccinated with Mycobacterium bovis (M. bovis) BCG with purified protein derivative of M. bovis (M. bovis PPD) induced 1α-hydroxylase gene expression and that treatment with a physiological concentration of 25(OH)D(3) down-regulated IFN-γ and IL-17F gene expression. Next, we stimulated PBMCs from M. bovis BCG-vaccinated and non-vaccinated cattle with M. bovis PPD and sorted them by FACS according to surface markers for monocytes/macrophages (CD14), B cells (IgM), and T cells (CD3). Sorting the PBMCs revealed that 1α-hydroxylase expression was induced in the monocytes and B cells, but not in the T cells. Furthermore, treatment of stimulated PBMCs with 25(OH)D(3) down-regulated antigen-specific IFN-γ and IL-17F responses in the T cells, even though 1α-hydroxylase expression was not induced in the T cells. Based on evidence of no T cell 1α-hydroxylase we hypothesize that activated monocytes and B cells synthesize 1,25(OH)(2)D(3) and that 1,25(OH)(2)D(3) down-regulates antigen-specific expression of IFN-γ and IL-17F in T cells in a paracrine fashion.     

T cell receptors employ diverse strategies to target a p53 cancer neoantigen

Adoptive cell therapy with tumor-specific T cells can mediate durable cancer regression. The prime target of tumor-specific T cells are neoantigens arising from mutations in self-proteins during malignant transformation. To understand T cell recognition of cancer neoantigens at the atomic level, we studied oligoclonal T cell receptors (TCRs) that recognize a neoepitope arising from a driver mutation in the p53 oncogene (p53R175H) presented by the major histocompatibility complex class I molecule HLA-A2. We previously reported the structures of three p53R175H-specific TCRs (38-10, 12-6, and 1a2) bound to p53R175H and HLA-A2. The structures showed that these TCRs discriminate between WT and mutant p53 by forming extensive interactions with the R175H mutation. Here, we report the structure of a fourth p53R175H-specific TCR (6-11) in complex with p53R175H and HLA-A2. In contrast to 38-10, 12-6, and 1a2, TCR 6-11 makes no direct contacts with the R175H mutation, yet is still able to distinguish mutant from WT p53. Structure-based in silico mutagenesis revealed that the 60-fold loss in 6-11 binding affinity for WT p53 compared to p53R175H is mainly due to the higher energetic cost of desolvating R175 in the WT p53 peptide during complex formation than H175 in the mutant. This indirect strategy for preferential neoantigen recognition by 6-11 is fundamentally different from the direct strategies employed by other TCRs and highlights the multiplicity of solutions to recognizing p53R175H with sufficient selectivity to mediate T cell killing of tumor but not normal cells.     

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.     

Characterization of mutations in the GTP-binding domain of IF2 resulting in cold-sensitive growth of Escherichia coli

The infB gene encodes translation initiation factor IF2. We have determined the entire sequence of infB from two cold-sensitive Escherichia coli strains IQ489 and IQ490. These two strains have been isolated as suppressor strains for the temperature-sensitive secretion mutation secY24. The mutations causing the suppression phenotype are located within infB. The only variations from the wild-type (wt) infB found in the two mutant strains are a replacement of Asp409 with Glu in strain IQ489 and an insertion of Gly between Ala421 and Gly422 in strain IQ490. Both positions are located in the GTP-binding G-domain of IF2. A model of the G-domain of E.coli IF2 is presented in. Physiological quantities of the recombinant mutant proteins were expressed in vivo in E.coli strains from which the chromosomal infB gene has been inactivated. At 42 degrees C, the mutants sustained normal cell growth, whereas a significant decrease in growth rate was found at 25 degrees C for both mutants as compared to wt IF2 expressed in the control strain. Circular dichroism spectra were recorded of the wt and the two mutant proteins to investigate the structural properties of the proteins. The spectra are characteristic of alpha-helix dominated structure, and reveal a significant different behavior between the wt and mutant IF2s with respect to temperature-induced conformational changes. The temperature-induced conformational change of the wt IF2 is a two-state process. In a ribosome-dependent GTPase assay in vitro the two mutants showed practically no activity at temperatures below 10 degrees C and a reduced activity at all temperatures up to 45 degrees C, as compared to wt IF2. The results indicate that the amino acid residues, Asp409 and Gly422, are located in important regions of the IF2 G-domain and demonstrate the importance of GTP hydrolysis in translation initiation for optimal cell growth.     

A recurrent neural model for proto-object based contour integration and figure-ground segregation

Visual processing of objects makes use of both feedforward and feedback streams of information. However, the nature of feedback signals is largely unknown, as is the identity of the neuronal populations in lower visual areas that receive them. Here, we develop a recurrent neural model to address these questions in the context of contour integration and figure-ground segregation. A key feature of our model is the use of grouping neurons whose activity represents tentative objects (“proto-objects”) based on the integration of local feature information. Grouping neurons receive input from an organized set of local feature neurons, and project modulatory feedback to those same neurons. Additionally, inhibition at both the local feature level and the object representation level biases the interpretation of the visual scene in agreement with principles from Gestalt psychology. Our model explains several sets of neurophysiological results (Zhou et al. Journal of Neuroscience, 20(17), 6594–6611 2000; Qiu et al. Nature Neuroscience, 10(11), 1492–1499 2007; Chen et al. Neuron, 82(3), 682–694 2014), and makes testable predictions about the influence of neuronal feedback and attentional selection on neural responses across different visual areas. Our model also provides a framework for understanding how object-based attention is able to select both objects and the features associated with them.     

Latitudinal patterns of range size and species richness of New World woody plants

Aim  Relationships between range size and species richness are contentious, yet they are key to testing the various hypotheses that attempt to explain latitudinal diversity gradients. Our goal is to utilize the largest data set yet compiled for New World woody plant biogeography to describe and assess these relationships between species richness and range size.
Location  North and South America.
Methods  We estimated the latitudinal extent of 12,980 species of woody plants (trees, shrubs, lianas). From these estimates we quantified latitudinal patterns of species richness and range size. We compared our observations with expectations derived from two null models.
Results   Peak richness and the smallest- and largest-ranged species are generally found close to the equator. In contrast to prominent diversity hypotheses: (1) mean latitudinal extent of tropical species is greater than expected; (2) latitudinal extent appears to be decoupled from species richness across New World latitudes, with abrupt transitions across subtropical latitudes; and (3) mean latitudinal extents show equatorial and north temperate peaks and subtropical minima. Our results suggest that patterns of range size and richness appear to be influenced by three broadly overlapping biotic domains (biotic provinces) for New World woody plants.
Main conclusions  Hypotheses that assume a direct relationship between range size and species richness may explain richness patterns within these domains, but cannot explain gradients in richness across the New World.     

Sensitivity of yeast strains with long G-tails to levels of telomere-bound telomerase

The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation.     

Drug block of the hERG potassium channel: insight from modeling

Many commonly used, structurally diverse, drugs block the human ether-a-go-go-related gene (hERG) K(+) channel to cause acquired long QT syndrome, which can lead to sudden death via lethal cardiac arrhythmias. This undesirable side effect is a major hurdle in the development of safe drugs. To gain insight about the structure of hERG and the nature of drug block we have produced structural models of the channel pore domain, into each of which we have docked a set of 20 hERG blockers. In the absence of an experimentally determined three-dimensional structure of hERG, each of the models was validated against site-directed mutagenesis data. First, hERG models were produced of the open and closed channel states, based on homology with the prokaryotic K(+) channel crystal structures. The modeled complexes were in partial agreement with the mutagenesis data. To improve agreement with mutagenesis data, a KcsA-based model was refined by rotating the four copies of the S6 transmembrane helix half a residue position toward the C-terminus, so as to place all residues known to be involved in drug binding in positions lining the central cavity. This model produces complexes that are consistent with mutagenesis data for smaller, but not larger, ligands. Larger ligands could be accommodated following refinement of this model by enlarging the cavity using the inherent flexibility about the glycine hinge (Gly648) in S6, to produce results consistent with the experimental data for the majority of ligands tested.