Integrating the Genetic and Physical Maps of Arabidopsis thaliana: Identification of Mapped Alleles of Cloned Essential (EMB) Genes

The classical genetic map of Arabidopsis includes more than 130 genes with an embryo-defective (emb) mutant phenotype. Many of these essential genes remain to be cloned. Hundreds of additional EMB genes have been cloned and catalogued (www.seedgenes.org) but not mapped. To facilitate EMB gene identification and assess the current level of saturation, we updated the classical map, compared the physical and genetic locations of mapped loci, and performed allelism tests between mapped (but not cloned) and cloned (but not mapped) emb mutants with similar chromosome locations. Two hundred pairwise combinations of genes located on chromosomes 1 and 5 were tested and more than 1100 total crosses were screened. Sixteen of 51 mapped emb mutants examined were found to be disrupted in a known EMB gene. Alleles of a wide range of published EMB genes (YDA, GLA1, TIL1, AtASP38, AtDEK1, EMB506, DG1, OEP80) were discovered. Two EMS mutants isolated 30 years ago, T-DNA mutants with complex insertion sites, and a mutant with an atypical, embryo-specific phenotype were resolved. The frequency of allelism encountered was consistent with past estimates of 500 to 1000 EMB loci. New EMB genes identified among mapped T-DNA insertion mutants included CHC1, which is required for chromatin remodeling, and SHS1/AtBT1, which encodes a plastidial nucleotide transporter similar to the maize Brittle1 protein required for normal endosperm development. Two classical genetic markers (PY, ALB1) were identified based on similar map locations of known genes required for thiamine (THIC) and chlorophyll (PDE166) biosynthesis. The alignment of genetic and physical maps presented here should facilitate the continued analysis of essential genes in Arabidopsis and further characterization of a broad spectrum of mutant phenotypes in a model plant

Does the early frog catch the worm? Disentangling potential drivers of a parasite age–intensity relationship in tadpoles

The manner in which parasite intensity and aggregation varies with host age can provide insights into parasite dynamics and help identify potential means of controlling infections in humans and wildlife. A significant challenge is to distinguish among competing mechanistic hypotheses for the relationship between age and parasite intensity or aggregation. Because different mechanisms can generate similar relationships, testing among competing hypotheses can be difficult, particularly in wildlife hosts, and often requires a combination of experimental and model fitting approaches. We used field data, experiments, and model fitting to distinguish among ten plausible drivers of a curvilinear age–intensity relationship and increasing aggregation with host age for echinostome trematode infections of green frogs. We found little support for most of these proposed drivers but did find that the parsimonious explanation for the observed age–intensity relationship was seasonal exposure to echinostomes. The parsimonious explanation for the aggregated distribution of parasites in this host population was heterogeneity in exposure. A predictive model incorporating seasonal exposure indicated that tadpoles hatching early or late in the breeding season should have lower trematode burdens at metamorphosis, particularly with simulated warmer climates. Application of this multi-pronged approach (field surveys, lab experiments, and modeling) to additional parasite–host systems could lead to discovery of general patterns in the drivers of parasite age–intensity and age–distribution relationships

GBR 12909 administration as a mouse model of bipolar disorder mania: mimicking quantitative assessment of manic behavior

Mania is a core feature of bipolar disorder (BD) that traditionally is assessed using rating scales. Studies using a new human behavioral pattern monitor (BPM) recently demonstrated that manic BD patients exhibit a specific profile of behavior that differs from schizophrenia and is characterized by increased motor activity, increased specific exploration, and perseverative locomotor patterns as assessed by spatial d. It was hypothesized that disrupting dopaminergic homeostasis by inhibiting dopamine transporter (DAT) function would produce a BD mania-like phenotype in mice as assessed by the mouse BPM. We compared the spontaneous locomotor and exploratory behavior of C57BL/6J mice treated with the catecholamine transporter inhibitor amphetamine or the selective DAT inhibitor GBR 12909 in the mouse BPM. We also assessed the duration of the effect of GBR 12909 by testing mice in the BPM for 3 h and its potential strain dependency by testing 129/SvJ mice. Amphetamine produced hyperactivity and increased perseverative patterns of locomotion as reflected in reduced spatial d values but reduced exploratory activity in contrast to the increased exploration observed in BD patients. GBR 12909 increased activity and reduced spatial d in combination with increased exploratory behavior, irrespective of inbred strain. These effects persisted for at least 3 h. Thus, selectively inhibiting the DAT produced a long-lasting cross-strain behavioral profile in mice that was consistent with that observed in manic BD patients. These findings support the use of selective DAT inhibition in animal models of the impaired dopaminergic homeostasis putatively involved in the pathophysiology of BD mania

Mapping Protein Interactions between Dengue Virus and Its Human and Insect Hosts

Dengue virus (DENV) represents a major disease burden in tropical and subtropical regions of the world, and has shown an increase in the number of cases in recent years. DENV is transmitted to humans through the bite of an infected mosquito, typically Aedes aegypti, after which it begins the infection and replication lifecycle within human cells. To perform the molecular functions required for invasion, replication, and spread of the virus, proteins encoded by DENV must interact with and alter the behavior of protein networks in both of these hosts. In this work, we used a computational method based on protein structures to predict interactions between DENV and its human and insect hosts. We predict numerous interactions, with many involved in known cell death, stress, and immune system pathways. Further investigation of these predicted protein-protein interactions should provide targets to combat the clinical manifestations of this disease in humans as well as points of intervention focused within the mosquito vector

Penetration of the Stigma and Style Elicits a Novel Transcriptome in Pollen Tubes, Pointing to Genes Critical for Growth in a Pistil

Pollen tubes extend through pistil tissues and are guided to ovules where they release sperm for fertilization. Although pollen tubes can germinate and elongate in a synthetic medium, their trajectory is random and their growth rates are slower compared to growth in pistil tissues. Furthermore, interaction with the pistil renders pollen tubes competent to respond to guidance cues secreted by specialized cells within the ovule. The molecular basis for this potentiation of the pollen tube by the pistil remains uncharacterized. Using microarray analysis in Arabidopsis, we show that pollen tubes that have grown through stigma and style tissues of a pistil have a distinct gene expression profile and express a substantially larger fraction of the Arabidopsis genome than pollen grains or pollen tubes grown in vitro. Genes involved in signal transduction, transcription, and pollen tube growth are overrepresented in the subset of the Arabidopsis genome that is enriched in pistil-interacted pollen tubes, suggesting the possibility of a regulatory network that orchestrates gene expression as pollen tubes migrate through the pistil. Reverse genetic analysis of genes induced during pollen tube growth identified seven that had not previously been implicated in pollen tube growth. Two genes are required for pollen tube navigation through the pistil, and five genes are required for optimal pollen tube elongation in vitro. Our studies form the foundation for functional genomic analysis of the interactions between the pollen tube and the pistil, which is an excellent system for elucidation of novel modes of cell–cell interaction

ICAR: endoscopic skull‐base surgery

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Structural and functional analysis of cell wall-anchored polypeptide adhesin BspA in <em>Streptococcus agalactiae</em>

\ua9 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Streptococcus agalactiae (group B Streptococcus, GBS) is the predominant cause of early-onset infectious disease in neonates and is responsible for life-threatening infections in elderly and immunocompromised individuals. Clinical manifestations of GBS infection include sepsis, pneumonia, and meningitis. Here, we describe BspA, a deviant antigen I/II family polypeptide that confers adhesive properties linked to pathogenesis in GBS. Heterologous expression of BspA on the surface of the non-adherent bacterium Lactococcus lactis confers adherence to scavenger receptor gp340, human vaginal epithelium, and to the fungus Candida albicans. Complementary crystallographic and biophysical characterization of BspA reveal a novel →-sandwich adhesion domain and unique asparagine-dependent super-helical stalk. Collectively, these findings establish a new bacterial adhesin structure that has in effect been hijacked by a pathogenic Streptococcus species to provide competitive advantage in human mucosal infections

Brazilin Removes Toxic Alpha-Synuclein and Seeding Competent Assemblies from Parkinson Brain by Altering Conformational Equilibrium

Alpha-synuclein (α-syn) fibrils, a major constituent of the neurotoxic Lewy Bodies in Parkinson’s disease, form via nucleation dependent polymerization and can replicate by a seeding mechanism. Brazilin, a small molecule derived from red cedarwood trees in Brazil, has been shown to inhibit the fibrillogenesis of amyloid-beta (Aβ) and α-syn as well as remodel mature fibrils and reduce cytotoxicity. Here we test the effects of Brazilin on both seeded and unseeded α-syn fibril formation and show that the natural polyphenol inhibits fibrillogenesis of α-syn by a unique mechanism that alters conformational equilibria in two separate points of the assembly mechanism: Brazilin preserves the natively unfolded state of α-syn by specifically binding to the compact conformation of the α-syn monomer. Brazilin also eliminates seeding competence of α-syn assemblies from Parkinson’s disease patient brain tissue, and reduces toxicity of pre-formed assemblies in primary neurons by inducing the formation of large fibril clusters. Molecular docking of Brazilin shows the molecule to interact both with unfolded α-syn monomers and with the cross-β sheet structure of α-syn fibrils. Our findings suggest that Brazilin has substantial potential as a neuroprotective and therapeutic agent for Parkinson’s disease

The type 2 diabetes gene product STARD10 is a phosphoinositide-binding protein that controls insulin secretory granule biogenesis

OBJECTIVE: Risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in the β-cell, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios. Although likely to serve as a mediator of intracellular lipid transfer, the identity of the transported lipids and thus the pathways through which STARD10 regulates β-cell function are not understood. The aim of this study was to identify the lipids transported and affected by STARD10 in the β-cell and the role of the protein in controlling proinsulin processing and insulin granule biogenesis and maturation. METHODS: We used isolated islets from mice deleted selectively in the β-cell for Stard10 (βStard10KO) and performed electron microscopy, pulse-chase, RNA sequencing, and lipidomic analyses. Proteomic analysis of STARD10 binding partners was executed in the INS1 (832/13) cell line. X-ray crystallography followed by molecular docking and lipid overlay assay was performed on purified STARD10 protein. RESULTS: βStard10KO islets had a sharply altered dense core granule appearance, with a dramatic increase in the number of "rod-like" dense cores. Correspondingly, basal secretion of proinsulin was increased versus wild-type islets. The solution of the crystal structure of STARD10 to 2.3 Å resolution revealed a binding pocket capable of accommodating polyphosphoinositides, and STARD10 was shown to bind to inositides phosphorylated at the 3' position. Lipidomic analysis of âStard10KO islets demonstrated changes in phosphatidylinositol levels, and the inositol lipid kinase PIP4K2C was identified as a STARD10 binding partner. Also consistent with roles for STARD10 in phosphoinositide signalling, the phosphoinositide-binding proteins Pirt and Synaptotagmin 1 were amongst the differentially expressed genes in βStard10KO islets. CONCLUSION: Our data indicate that STARD10 binds to, and may transport, phosphatidylinositides, influencing membrane lipid composition, insulin granule biosynthesis, and insulin processing