Resolving Relationships at the Animal-Fungal Divergence: A Molecular Phylogenetic Approach for Understanding the Ecology and Evolution of the Protist Trichomycetes

The Ichthyosporea (= Mesomycetozoea) is a relatively understudied class of unicellular symbionts that molecular phylogenies have placed at the divergence of animals and fungi. Subsumed in this class are the cosmopolitan families Eccrinidae and Amoebidiidae (referred to as “protist trichos” or “trichos” herein), which are considered obligate commensal endobionts of various arthropods, including marine, freshwater and terrestrial hosts. Once thought to be members of the fungal class Trichomycetes due to their hyphal-like growth form and ecological similarity, molecular evidence has necessitated reclassification. However, evolutionary relationships within and between them are still unclear as the number of taxa sampled and/or the amount of gene data gathered have been factors limiting resolution. These organisms are also taxonomically challenging since informative, homologous morphological characters are difficult to discern using only a light microscope (the method by which members of Amoebidiidae and Eccrinidae have traditionally been described), and only a few have been obtained in axenic culture. Most protist trichos reported thus far lack sufficiently detailed morphological parameters to permit ease and confidence in species identification. As such, relatively little is known about the ecology and biology of most members, some of which were originally classified as fungi or algae. As new members were discovered or reclassified, two orders were established: Dermocystida and Eccrinida. Whereas members of the Dermocystida are almost entirely parasites of various metazoan hosts, only three clades within the Eccrinida contain known parasites, with the remaining members regarded as commensalistic. Interestingly, the putative closest extant relative to both groups is Ichthyophonus, an economically relevant fish parasite, which can invade vital host tissues (e.g. heart and liver) via circulating amoeba-like cells,causing disease and potentially death. The most recent molecular systematic study of the protist trichos was published about a decade ago, and there is as yet but one Paramoebidium (Amoebidiidae) sequence deposited in GenBank. Currently, based on molecular data, the Amoebidiidae are supported as monophyletic (based on one sample from each of its two genera) while the monophyly of the Eccrinidae is indicated, but not supported. Likewise, the relationship of the protist trichos to Ichthyophonus remains unresolved. As such, the first chapter of this thesis addressed the molecular phylogeny of order Eccrinida, with particular emphasis on the protist trichos by first amplifying and sequencing rDNA genes (18S and 28S) for over 100 new samples. Amplification tests were also attempted for several protein-coding genes, including heat shock protein 70. The resulting tree inferences were used in subsequent analyses of ecological and life history traits via ancestral state reconstructions and Bayesian tip-association significance testing (BaTS). In the second chapter, samples of Paramoebidium spp. were morphologically and molecularly assessed as a case study into the utility of traditionally described morphological characters for taxonomic delimitation among protist trichos. Morphological differentiation of Paramoebidium spp. has been notoriously problematic due to inter- and intraspecific variability. Host specificity within the genus was early suggested, but later questioned, and has not been subjected to thorough evaluation. Therefore, host and hyphal characters were analyzed via three different methods of ancestral state reconstruction, as well as with BaTS on a molecular phylogeny of over 70 Amoebidiidae samples. Results of these studies indicate: 1) contrary to previous hypotheses, the Amoebidiidae may be paraphyletic, 2) relationships among Eccrinidae and between the protist trichos and Ichthyophonus remain unresolved, 3) several life history and host characters are significantly associated with both the Eccrinida and Amoebidiidae phylogenies, providing platforms for future hypothesis formulation, 4) the protist trichos and the Eccrinida as a whole are likely much more species rich and widespread than what is currently known, 5) species delimitation within Paramoebidium is complicated by cryptic speciation, but there is evidence for possible host specificity, and 6) future studies of the protist trichos will benefit from an integrated approach that shifts away from an emphasis on the morphological species concept but includes both genetic sequence data and traditional morphological approaches

Characterization of the 1st and 2nd EF-hands of NADPH oxidase 5 by fluorescence, isothermal titration calorimetry, and circular dichroism

<p>Abstract</p> <p>Background</p> <p>Superoxide generated by non-phagocytic NADPH oxidases (NOXs) is of growing importance for physiology and pathobiology. The calcium binding domain (CaBD) of NOX5 contains four EF-hands, each binding one calcium ion. To better understand the metal binding properties of the 1^stand 2^ndEF-hands, we characterized the N-terminal half of CaBD (NCaBD) and its calcium-binding knockout mutants.</p> <p>Results</p> <p>The isothermal titration calorimetry measurement for NCaBD reveals that the calcium binding of two EF-hands are loosely associated with each other and can be treated as independent binding events. However, the Ca²⁺binding studies on NCaBD(E31Q) and NCaBD(E63Q) showed their binding constants to be 6.5 × 10⁵and 5.0 × 10²M^-1with ΔHs of -14 and -4 kJ/mol, respectively, suggesting that intrinsic calcium binding for the 1^stnon-canonical EF-hand is largely enhanced by the binding of Ca²⁺to the 2^ndcanonical EF-hand. The fluorescence quenching and CD spectra support a conformational change upon Ca²⁺binding, which changes Trp residues toward a more non-polar and exposed environment and also increases its α-helix secondary structure content. All measurements exclude Mg²⁺-binding in NCaBD.</p> <p>Conclusions</p> <p>We demonstrated that the 1^stnon-canonical EF-hand of NOX5 has very weak Ca²⁺binding affinity compared with the 2^ndcanonical EF-hand. Both EF-hands interact with each other in a cooperative manner to enhance their Ca²⁺binding affinity. Our characterization reveals that the two EF-hands in the N-terminal NOX5 are Ca²⁺specific.</p> <p>Graphical abstract</p> <p><display-formula><graphic file="1752-153X-6-29-i1.gif"/></display-formula></p

A Day in the Life of Microcystis aeruginosa Strain PCC 7806 as Revealed by a Transcriptomic Analysis

The cyanobacterium, Microcystis aeruginosa, is able to proliferate in a wide range of freshwater ecosystems and to produce many secondary metabolites that are a threat to human and animal health. The dynamic of this production and more globally the metabolism of this species is still poorly known. A DNA microarray based on the genome of M. aeruginosa PCC 7806 was constructed and used to study the dynamics of gene expression in this cyanobacterium during the light/dark cycle, because light is a critical factor for this species, like for other photosynthetic microorganisms. This first application of transcriptomics to a Microcystis species has revealed that more than 25% of the genes displayed significant changes in their transcript abundance during the light/dark cycle and in particular during the dark/light transition. The metabolism of M. aeruginosa is compartmentalized between the light period, during which carbon uptake, photosynthesis and the reductive pentose phosphate pathway lead to the synthesis of glycogen, and the dark period, during which glycogen degradation, the oxidative pentose phosphate pathway, the TCA branched pathway and ammonium uptake promote amino acid biosynthesis. We also show that the biosynthesis of secondary metabolites, such as microcystins, aeruginosin and cyanopeptolin, occur essentially during the light period, suggesting that these metabolites may interact with the diurnal part of the central metabolism

Stable Mutated tau441 Transfected SH-SY5Y Cells as Screening Tool for Alzheimer’s Disease Drug Candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies

Targeted p120-Catenin Ablation Disrupts Dental Enamel Development

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows

A Novel Molecular Solution for Ultraviolet Light Detection in Caenorhabditis elegans

For many organisms the ability to transduce light into cellular signals is crucial for survival. Light stimulates DNA repair and metabolism changes in bacteria, avoidance responses in single-cell organisms, attraction responses in plants, and both visual and nonvisual perception in animals. Despite these widely differing responses, in all of nature there are only six known families of proteins that can transduce light. Although the roundworm Caenorhabditis elegans has none of the known light transduction systems, we show here that C. elegans strongly accelerates its locomotion in response to blue or shorter wavelengths of light, with maximal responsiveness to ultraviolet light. Our data suggest that C. elegans uses this light response to escape the lethal doses of sunlight that permeate its habitat. Short-wavelength light drives locomotion by bypassing two critical signals, cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG), that neurons use to shape and control behaviors. C. elegans mutants lacking these signals are paralyzed and unresponsive to harsh physical stimuli in ambient light, but short-wavelength light rapidly rescues their paralysis and restores normal levels of coordinated locomotion. This light response is mediated by LITE-1, a novel ultraviolet light receptor that acts in neurons and is a member of the invertebrate Gustatory receptor (Gr) family. Heterologous expression of the receptor in muscle cells is sufficient to confer light responsiveness on cells that are normally unresponsive to light. Our results reveal a novel molecular solution for ultraviolet light detection and an unusual sensory modality in C. elegans that is unlike any previously described light response in any organism

A Patient-Specific in silico Model of Inflammation and Healing Tested in Acute Vocal Fold Injury

The development of personalized medicine is a primary objective of the medical community and increasingly also of funding and registration agencies. Modeling is generally perceived as a key enabling tool to target this goal. Agent-Based Models (ABMs) have previously been used to simulate inflammation at various scales up to the whole-organism level. We extended this approach to the case of a novel, patient-specific ABM that we generated for vocal fold inflammation, with the ultimate goal of identifying individually optimized treatments. ABM simulations reproduced trajectories of inflammatory mediators in laryngeal secretions of individuals subjected to experimental phonotrauma up to 4 hrs post-injury, and predicted the levels of inflammatory mediators 24 hrs post-injury. Subject-specific simulations also predicted different outcomes from behavioral treatment regimens to which subjects had not been exposed. We propose that this translational application of computational modeling could be used to design patient-specific therapies for the larynx, and will serve as a paradigm for future extension to other clinical domains