New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes)

Digeneans and cestodes are species‐rich taxa and can seriously impact human health, fisheries, aqua‐ and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for species detection and identification, but both ‘universal’ and taxon‐specific COI primers fail to amplify in many flatworm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases.     

Age‐dependent alterations of the NMDA receptor developmental profile and adult behavior in postnatally ketamine‐treated mice

Ketamine is a NMDA receptor (NMDAR) antagonist used in pediatric anesthesia. Given the role of glutamatergic signaling during brain maturation, we studied the effects of a single ketamine injection (40 mg/kg s.c) in mouse neonates depending on postnatal age at injection (P2, P5, or P10) on cortical NMDAR subunits expression and association with Membrane‐Associated Guanylate Kinases PSD95 and SAP102. The effects of ketamine injection at P2, P5, or P10 on motor activity were compared in adulthood. Ketamine increased GluN2A and GluN2B mRNA levels in P2‐treated mice without change in proteins, while it decreased GluN2B protein in P10‐treated mice without change in mRNA. Ketamine reduced GluN2A mRNA and protein levels in P5‐treated mice without change in GluN2B and GluN1. Ketamine affected the GluN2A/PSD95 association regardless of the age at injection, while GluN2B/PSD95 association was enhanced only in P5‐treated mice. Microdissection of ketamine‐treated mouse cortex showed a decrease in GluN2A mRNA level in superficial layers (I–IV) and an increase in all subunit expressions in deep layers (V–VI) in P5‐ and P10‐treated mice, respectively. Our data suggest that ketamine impairs cortical NMDAR subunit developmental profile and delays the synaptic targeting of GluN2A‐enriched NMDAR. Ketamine injection at P2 or P10 resulted in hyperlocomotion in adult male mice in an open field, without change in females. Voluntary running‐wheel exercise showed age‐ and sex‐dependent alterations of the mouse activity, especially during the dark phase. Overall, a single neonatal ketamine exposure led to short‐term NMDAR cortical developmental profile impairments and long‐term motor activity alterations persisting in adulthood. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 315–333, 2015     

Alix is required for activity-dependent bulk endocytosis at brain synapses

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activity-dependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrin-independent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations.

The adaptor protein Alix (PDCD6IP) is necessary for membrane shaping underlying various biological processes including endocytosis. This study shows that Alix mediates activity-dependent bulk endocytosis and is required for correct synaptic physiology under normal and pathological conditions.     

A Bayesian framework for inference of the genotype-phenotype map for segregating populations

Complex genetic interactions lie at the foundation of many diseases. Understanding the nature of these interactions is critical to developing rational intervention strategies. In mammalian systems hypothesis testing in vivo is expensive, time consuming, and often restricted to a few physiological endpoints. Thus, computational methods that generate causal hypotheses can help to prioritize targets for experimental intervention. We propose a Bayesian statistical method to infer networks of causal relationships among genotypes and phenotypes using expression quantitative trait loci (eQTL) data from genetically randomized populations. Causal relationships between network variables are described with hierarchical regression models. Prior distributions on the network structure enforce graph sparsity and have the potential to encode prior biological knowledge about the network. An efficient Monte Carlo method is used to search across the model space and sample highly probable networks. The result is an ensemble of networks that provide a measure of confidence in the estimated network topology. These networks can be used to make predictions of system-wide response to perturbations. We applied our method to kidney gene expression data from an MRL/MpJ × SM/J intercross population and predicted a previously uncharacterized feedback loop in the local renin-angiotensin system.     

Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail.

We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 microM somatostatin (SST)-14 x 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.     

Long-term effects of irradiation with iron-56 particles on the nigrostriatal dopamine system

Exposure to heavy ions during a Mars mission might damage the brain, thus compromising mission success and the quality of life of returning astronauts. Several workers have suggested that the dopamine system is particularly sensitive to heavy ion radiation, but direct evidence for this notion is lacking. We examined measures of brain dopamine viability at times up to 15 months after acute exposure of rats to ⁵⁶Fe (1.2–2.4 Gy). No effects were seen in brain sections stained for tyrosine hydroxylase, the classical marker for dopamine cells and nerve terminals. Locomotion stimulated by cocaine, which directly activates the dopamine system, was reduced at 6 months but not at 12 months. Furthermore, in a visually cued lever-pressing test, reaction times, which are prolonged by dopamine system damage, were identical in irradiated and control animals. However, learning times were increased by irradiation. Our data suggest that the midbrain dopamine system is not especially sensitive to damage by ⁵⁶Fe particles at doses much higher than would be associated with travel to and from Mars.     

Halotolerant laccases from Chaetomium sp., Xylogone sphaerospora, and Coprinopsis sp. isolated from a Mediterranean coastal area

Laccases (EC 1.10.3.2) are phenoloxidases involved in the transformation of the recalcitrant fraction of organic matter in soil. These enzymes are also able to transform certain aromatic pollutants such as polycyclic aromatic hydrocarbons (PAHs) and are known to be inhibited by chloride ions. This study aims to test the potential of some fungal strains newly isolated from natural environments subjected to high osmotic pressure such as coastal ecosystems, to produce chloride tolerant laccases. Three strains were identified as Chaetomium sp., Xylogone sphaerospora (two Ascomycota), and Coprinopsis sp. (a Basidiomycota) and the laccases produced by these fungi were weakly inhibited by chloride ions compared with previous data from literature. Moreover, we tested their reactivity towards various PAHs which are widespread anthropic pollutants. They were able to transform anthracene to 9,10-anthraquinone and we determine 7.5 eV as the threshold of ionization potential for PAH oxidation by these laccases.