Site-specific perturbations of alpha-synuclein fibril structure by the Parkinson's disease associated mutations A53T and E46K.

PMCID: PMC3591419This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Parkinson's disease (PD) is pathologically characterized by the presence of Lewy bodies (LBs) in dopaminergic neurons of the substantia nigra. These intracellular inclusions are largely composed of misfolded α-synuclein (AS), a neuronal protein that is abundant in the vertebrate brain. Point mutations in AS are associated with rare, early-onset forms of PD, although aggregation of the wild-type (WT) protein is observed in the more common sporadic forms of the disease. Here, we employed multidimensional solid-state NMR experiments to assess A53T and E46K mutant fibrils, in comparison to our recent description of WT AS fibrils. We made de novo chemical shift assignments for the mutants, and used these chemical shifts to empirically determine secondary structures. We observe significant perturbations in secondary structure throughout the fibril core for the E46K fibril, while the A53T fibril exhibits more localized perturbations near the mutation site. Overall, these results demonstrate that the secondary structure of A53T has some small differences from the WT and the secondary structure of E46K has significant differences, which may alter the overall structural arrangement of the fibrils

Do female association preferences predict the likelihood of reproduction?

Sexual selection acting on male traits through female mate choice is commonly inferred from female association preferences in dichotomous mate choice experiments. However, there are surprisingly few empirical demonstrations that such association preferences predict the likelihood of females reproducing with a particular male. This information is essential to confirm association preferences as good predictors of mate choice. We used green swordtails (<i>Xiphophorus helleri</i>) to test whether association preferences predict the likelihood of a female reproducing with a male. Females were tested for a preference for long- or short-sworded males in a standard dichotomous choice experiment and then allowed free access to either their preferred or non-preferred male. If females subsequently failed to produce fry, they were provided a second unfamiliar male with similar sword length to the first male. Females were more likely to reproduce with preferred than non-preferred males, but for those that reproduced, neither the status (preferred/non-preferred) nor the sword length (long/short) of the male had an effect on brood size or relative investment in growth by the female. There was no overall preference based on sword length in this study, but male sword length did affect likelihood of reproduction, with females more likely to reproduce with long- than short-sworded males (independent of preference for such males in earlier choice tests). These results suggest that female association preferences are good indicators of female mate choice but that ornament characteristics of the male are also important

Defective Peripheral Nerve Development Is Linked to Abnormal Architecture and Metabolic Activity of Adipose Tissue in Nscl-2 Mutant Mice

BACKGROUND: In mammals the interplay between the peripheral nervous system (PNS) and adipose tissue is widely unexplored. We have employed mice, which develop an adult onset of obesity due to the lack the neuronal specific transcription factor Nscl-2 to investigate the interplay between the nervous system and white adipose tissue (WAT). METHODOLOGY: Changes in the architecture and innervation of WAT were compared between wildtype, Nscl2-/-, ob/ob and Nscl2-/-//ob/ob mice using morphological methods, immunohistochemistry and flow cytometry. Metabolic alterations in mutant mice and in isolated cells were investigated under basal and stimulated conditions. PRINCIPAL FINDINGS: We found that Nscl-2 mutant mice show a massive reduction of innervation of white epididymal and paired subcutaneous inguinal fat tissue including sensory and autonomic nerves as demonstrated by peripherin and neurofilament staining. Reduction of innervation went along with defects in the formation of the microvasculature, accumulation of cells of the macrophage/preadipocyte lineage, a bimodal distribution of the size of fat cells, and metabolic defects of isolated adipocytes. Despite a relative insulin resistance of white adipose tissue and isolated Nscl-2 mutant adipocytes the serum level of insulin in Nscl-2 mutant mice was only slightly increased. CONCLUSIONS: We conclude that the reduction of the innervation and vascularization of WAT in Nscl-2 mutant mice leads to the increase of preadipocyte/macrophage-like cells, a bimodal distribution of the size of adipocytes in WAT and an altered metabolic activity of adipocytes

Comparative analysis of miRNAs and their targets across four plant species

BACKGROUND: MicroRNA (miRNA) mediated regulation of gene expression has been recognized as a major posttranscriptional regulatory mechanism also in plants. We performed a comparative analysis of miRNAs and their respective gene targets across four plant species: Arabidopsis thaliana (Ath), Medicago truncatula(Mtr), Brassica napus (Bna), and Chlamydomonas reinhardtii (Cre). RESULTS: miRNAs were obtained from mirBase with 218 miRNAs for Ath, 375 for Mtr, 46 for Bna, and 73 for Cre, annotated for each species respectively. miRNA targets were obtained from available database annotations, bioinformatic predictions using RNAhybrid as well as predicted from an analysis of mRNA degradation products (degradome sequencing) aimed at identifying miRNA cleavage products. On average, and considering both experimental and bioinformatic predictions together, every miRNA was associated with about 46 unique gene transcripts with considerably variation across species. We observed a positive and linear correlation between the number miRNAs and the total number of transcripts across different plant species suggesting that the repertoire of miRNAs correlates with the size of the transcriptome of an organism. Conserved miRNA-target pairs were found to be associated with developmental processes and transcriptional regulation, while species-specific (in particular, Ath) pairs are involved in signal transduction and response to stress processes. Conserved miRNAs have more targets and higher expression values than non-conserved miRNAs. We found evidence for a conservation of not only the sequence of miRNAs, but their expression levels as well. CONCLUSIONS: Our results support the notion of a high birth and death rate of miRNAs and that miRNAs serve many species specific functions, while conserved miRNA are related mainly to developmental processes and transcriptional regulation with conservation operating at both the sequence and expression level

Electron Antineutrino Search at the Sudbury Neutrino Observatory

Upper limits on the \nuebar flux at the Sudbury Neutrino Observatory have been set based on the \nuebar charged-current reaction on deuterium. The reaction produces a positron and two neutrons in coincidence. This distinctive signature allows a search with very low background for \nuebar's from the Sun and other potential sources. Both differential and integral limits on the \nuebar flux have been placed in the energy range from 4 -- 14.8 MeV. For an energy-independent \nu_e --> \nuebar conversion mechanism, the integral limit on the flux of solar \nuebar's in the energy range from 4 -- 14.8 MeV is found to be \Phi_\nuebar <= 3.4 x 10^4 cm^{-2} s^{-1} (90% C.L.), which corresponds to 0.81% of the standard solar model 8B \nu_e flux of 5.05 x 10^6 cm^{-2} s^{-1}, and is consistent with the more sensitive limit from KamLAND in the 8.3 -- 14.8 MeV range of 3.7 x 10^2 cm^{-2} s^{-1} (90% C.L.). In the energy range from 4 -- 8 MeV, a search for \nuebar's is conducted using coincidences in which only the two neutrons are detected. Assuming a \nuebar spectrum for the neutron induced fission of naturally occurring elements, a flux limit of Phi_\nuebar <= 2.0 x 10^6 cm^{-2} s^{-1}(90% C.L.) is obtained.Comment: submitted to Phys. Rev.

Effect of Spermidine on Misfolding and Interactions of Alpha-Synuclein

Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation – misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn

CGRPα-Expressing Sensory Neurons Respond to Stimuli that Evoke Sensations of Pain and Itch

Calcitonin gene-related peptide (CGRPα, encoded by Calca) is a classic marker of nociceptive dorsal root ganglia (DRG) neurons. Despite years of research, it is unclear what stimuli these neurons detect in vitro or in vivo. To facilitate functional studies of these neurons, we genetically targeted an axonal tracer (farnesylated enhanced green fluorescent protein; GFP) and a LoxP-stopped cell ablation construct (human diphtheria toxin receptor; DTR) to the Calca locus. In culture, 10–50% (depending on ligand) of all CGRPα-GFP-positive (+) neurons responded to capsaicin, mustard oil, menthol, acidic pH, ATP, and pruritogens (histamine and chloroquine), suggesting a role for peptidergic neurons in detecting noxious stimuli and itch. In contrast, few (2.2±1.3%) CGRPα-GFP+ neurons responded to the TRPM8-selective cooling agent icilin. In adult mice, CGRPα-GFP+ cell bodies were located in the DRG, spinal cord (motor neurons and dorsal horn neurons), brain and thyroid—reproducibly marking all cell types known to express Calca. Half of all CGRPα-GFP+ DRG neurons expressed TRPV1, ∼25% expressed neurofilament-200, <10% contained nonpeptidergic markers (IB4 and Prostatic acid phosphatase) and almost none (<1%) expressed TRPM8. CGRPα-GFP+ neurons innervated the dorsal spinal cord and innervated cutaneous and visceral tissues. This included nerve endings in the epidermis and on guard hairs. Our study provides direct evidence that CGRPα+ DRG neurons respond to agonists that evoke pain and itch and constitute a sensory circuit that is largely distinct from nonpeptidergic circuits and TRPM8+/cool temperature circuits. In future studies, it should be possible to conditionally ablate CGRPα-expressing neurons to evaluate sensory and non-sensory functions for these neurons

microPIR: An Integrated Database of MicroRNA Target Sites within Human Promoter Sequences

Background: microRNAs are generally understood to regulate gene expression through binding to target sequences within 39-UTRs of mRNAs. Therefore, computational prediction of target sites is usually restricted to these gene regions. Recent experimental studies though have suggested that microRNAs may alternatively modulate gene expression by interacting with promoters. A database of potential microRNA target sites in promoters would stimulate research in this field leading to more understanding of complex microRNA regulatory mechanism. Methodology: We developed a database hosting predicted microRNA target sites located within human promoter sequences and their associated genomic features, called microPIR (microRNA-Promoter Interaction Resource). microRNA seed sequences were used to identify perfect complementary matching sequences in the human promoters and the potential target sites were predicted using the RNAhybrid program..15 million target sites were identified which are located within 5000 bp upstream of all human genes, on both sense and antisense strands. The experimentally confirmed argonaute (AGO) binding sites and EST expression data including the sequence conservation across vertebrate species of each predicted target are presented for researchers to appraise the quality of predicted target sites. The microPIR database integrates various annotated genomic sequence databases, e.g. repetitive elements, transcription factor binding sites, CpG islands, and SNPs, offering users the facility to extensively explore relationships among target sites and other genomi

Factors confounding the assessment of reflection: a critical review

BACKGROUND: Reflection on experience is an increasingly critical part of professional development and lifelong learning. There is, however, continuing uncertainty about how best to put principle into practice, particularly as regards assessment. This article explores those uncertainties in order to find practical ways of assessing reflection. DISCUSSION: We critically review four problems: 1. Inconsistent definitions of reflection; 2. Lack of standards to determine (in)adequate reflection; 3. Factors that complicate assessment; 4. Internal and external contextual factors affecting the assessment of reflection. SUMMARY: To address the problem of inconsistency, we identified processes that were common to a number of widely quoted theories and synthesised a model, which yielded six indicators that could be used in assessment instruments. We arrived at the conclusion that, until further progress has been made in defining standards, assessment must depend on developing and communicating local consensus between stakeholders (students, practitioners, teachers, supervisors, curriculum developers) about what is expected in exercises and formal tests. Major factors that complicate assessment are the subjective nature of reflection's content and the dependency on descriptions by persons being assessed about their reflection process, without any objective means of verification. To counter these validity threats, we suggest that assessment should focus on generic process skills rather than the subjective content of reflection and where possible to consider objective information about the triggering situation to verify described reflections. Finally, internal and external contextual factors such as motivation, instruction, character of assessment (formative or summative) and the ability of individual learning environments to stimulate reflection should be considered