Characterization and taxonomic validity of the ciliate Oxytricha trifallax (Class Spirotrichea) based on multiple gene sequences: limitations in identifying genera solely by morphology

Oxytricha trifallax - an established model organism for studying genome rearrangements, chromosome structure, scrambled genes, RNA-mediated epigenetic inheritance, and other phenomena - has been the subject of a nomenclature controversy for several years. Originally isolated as a sibling species of O. fallax, O. trifallax was reclassified in 1999 as Sterkiella histriomuscorum, a previously identified species, based on morphological similarity. The proper identification of O. trifallax is crucial to resolve in order to prevent confusion in both the comparative genomics and the general scientific communities. We analyzed nine conserved nuclear gene sequences between the two given species and several related ciliates. Phylogenetic analyses suggest that O. trifallax and a bona fide S. histriomuscorum have accumulated significant evolutionary divergence from each other relative to other ciliates such that they should be unequivocally classified as separate species. We also describe the original isolation of O. trifallax, including its comparison to O. fallax, and we provide criteria to identify future isolates of O. trifallax.     

Oxylipin and mitochondrion probes to track yeast sexual cells

When oxylipin and mitochondrion probes, i.e., fluorescing antibodies specific for 3-hydroxy fatty acids (3-OH oxylipins) and rhodamine 123 (Rh123), were added to yeast cells, these probes accumulated mainly in the sexual cells (i.e., both associated with ascospores) and not in the vegetative cells. This suggests increased mitochondrial activity in asci, since 3-OH oxylipins are mitochondrially produced and it is known that Rh123 accumulates selectively in functional mitochondria that maintain a high transmembrane potential (Delta Psi m). This increased activity may be necessary for the production and effective release of the many spores found in single-celled asci. These results may be useful in the rapid identification of asci and in yeast sexual spore mechanics, which may find application in yeast systematics as well as hydro-, aero-, and nano-technologies.     

An inducible caspase 9 safety switch can halt cell therapy-induced autoimmune disease

Transfer of either allogeneic or genetically modified T cells as a therapy for malignancies can be accompanied by T cell-mediated tissue destruction. The introduction of an efficient "safety switch" can potentially be used to control the survival of adoptively transferred cell populations and as such reduce the risk of severe graft-vs-host disease. In this study, we have tested the value of an inducible caspase 9-based safety switch to halt an ongoing immune attack in a murine model for cell therapy-induced type I diabetes. The data obtained in this model indicate that self-reactive T cells expressing this conditional safety switch show unimpaired lymphopenia- and vaccine-induced proliferation and effector function in vivo, but can be specifically and rapidly eliminated upon triggering. These data provide strong support for the evaluation of this conditional safety switch in clinical trials of adoptive cell therapy.     

Interaction of acetylcholinesterase with the G4 domain of the laminin alpha1-chain

Although the primary function of AChE (acetylcholinesterase) is the synaptic hydrolysis of acetylcholine, it appears that the protein is also able to promote various non-cholinergic activities, including cell adhesion, neurite outgrowth and amyloidosis. We have observed previously that AChE is able to bind to mouse laminin-111 in vitro by an electrostatic mechanism. We have also observed that certain mAbs (monoclonal antibodies) recognizing AChE's PAS (peripheral anionic site) inhibit both laminin binding and cell adhesion in neuroblastoma cells. Here, we investigated the interaction sites of the two molecules, using docking, synthetic peptides, ELISAs and conformational interaction site mapping. Mouse AChE was observed on docking to bind to a discontinuous, largely basic, structure, Val(2718)-Arg-Lys-Arg-Leu(2722), Tyr(2738)-Tyr(2739), Tyr(2789)-Ile-Lys-Arg-Lys(2793) and Val(2817)-Glu-Arg-Lys(2820), on the mouse laminin alpha1 G4 domain. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps extensively with laminin's heparin-binding site, and AChE was observed to compete with heparan sulfate for laminin binding. Docking showed the major component of the interaction site on AChE to be the acidic sequence Arg(90)-Glu-Leu-Ser-Glu-Asp(95) on the omega loop, and also the involvement of Pro(40)-Pro-Val(42), Arg(46) (linked to Glu(94) by a salt bridge) and the hexapeptide Asp(61)-Ala-Thr-Thr-Phe-Gln(66). Epitope analysis, using CLiPS technology, of seven adhesion-inhibiting mAbs (three anti-human AChE, one anti-Torpedo AChE and three anti-human anti-anti-idiotypic antibodies) showed their major recognition site to be the sequence Pro(40)-Pro-Met-Gly-Pro-Arg-Arg-Phe(48) (AChE human sequence). The antibodies, however, also reacted with the proline-containing sequences Pro(78)-Gly-Phe-Glu-Gly-Thr-Glu(84) and Pro(88)-Asn-Arg-Glu-Leu-Ser-Glu-Asp(95). Antibodies that recognized other features of the PAS area but not the Arg(90)-Gly-Leu-Ser-Glu-Asp(95) motif interfered neither with laminin binding nor with cell adhesion. These results define sites for the interaction of AChE and laminin and suggest that the interaction plays a role in cell adhesion. They also suggest the strong probability of functional redundancy between AChE and other molecules in early development, particularly heparan sulfate proteoglycans, which may explain the survival of the AChE-knockout mouse.     

Binets: Fundamental Building Blocks for Phylogenetic Networks

Phylogenetic networks are a generalization of evolutionary trees that are used by biologists to represent the evolution of organisms which have undergone reticulate evolution. Essentially, a phylogenetic network is a directed acyclic graph having a unique root in which the leaves are labelled by a given set of species. Recently, some approaches have been developed to construct phylogenetic networks from collections of networks on 2- and 3-leaved networks, which are known as binets and trinets, respectively. Here we study in more depth properties of collections of binets, one of the simplest possible types of networks into which a phylogenetic network can be decomposed. More specifically, we show that if a collection of level-1 binets is compatible with some binary network, then it is also compatible with a binary level-1 network. Our proofs are based on useful structural results concerning lowest stable ancestors in networks. In addition, we show that, although the binets do not determine the topology of the network, they do determine the number of reticulations in the network, which is one of its most important parameters. We also consider algorithmic questions concerning binets. We show that deciding whether an arbitrary set of binets is compatible with some network is at least as hard as the well-known graph isomorphism problem. However, if we restrict to level-1 binets, it is possible to decide in polynomial time whether there exists a binary network that displays all the binets. We also show that to find a network that displays a maximum number of the binets is NP-hard, but that there exists a simple polynomial-time 1/3-approximation algorithm for this problem. It is hoped that these results will eventually assist in the development of new methods for constructing phylogenetic networks from collections of smaller networks.     

Reduced connectivity in the self-processing network of schizophrenia patients with poor insight

Lack of insight (unawareness of illness) is a common and clinically relevant feature of schizophrenia. Reduced levels of self-referential processing have been proposed as a mechanism underlying poor insight. The default mode network (DMN) has been implicated as a key node in the circuit for self-referential processing. We hypothesized that during resting state the DMN network would show decreased connectivity in schizophrenia patients with poor insight compared to patients with good insight. Patients with schizophrenia were recruited from mental health care centers in the north of the Netherlands and categorized in groups having good insight (n = 25) or poor insight (n = 19). All subjects underwent a resting state fMRI scan. A healthy control group (n = 30) was used as a reference. Functional connectivity of the anterior and posterior part of the DMN, identified using Independent Component Analysis, was compared between groups. Patients with poor insight showed lower connectivity of the ACC within the anterior DMN component and precuneus within the posterior DMN component compared to patients with good insight. Connectivity between the anterior and posterior part of the DMN was lower in patients than controls, and qualitatively different between the good and poor insight patient groups. As predicted, subjects with poor insight in psychosis showed decreased connectivity in DMN regions implicated in self-referential processing, although this concerned only part of the network. This finding is compatible with theories implying a role of reduced self-referential processing as a mechanism contributing to poor insight.     

Boltzmann relation reliability in optical temperature sensing based on upconversion studies of Er3+/Yb3+ co-doped PZT ceramics

The fluorescence intensity ratio (FIR) of two thermally coupled levels with temperature follows the Boltzmann equation and shows an exponential nature to the temperature that is purely dependent on the energy difference between the levels. Despite the identical energy difference between the thermally coupled levels, researchers have observed varying sensitivities for various samples. In this article, the FIR and sensitivities were calculated using the Boltzmann equation by changing various parameters such as energy difference (ΔE) and the value of the constant C. The results were compared with various reports for Er³⁺/Yb³⁺ ions. After analysis, a new polynomial fit equation was used to determine the temperature sensitivities for the Er³⁺/Yb³⁺ co-doped PbZrTiO₃ phosphor in lieu of the conventional Boltzmann equation. The polynomial fit equation eliminated the dependency of the sensitivity on the inverse of the FIR factor and a flat sensitivity curve was obtained with temperature.