Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

Development of SCAR markers linked to a gene controlling absence of tannins in faba bean

Faba beans are inexpensive, nutrient-dense sources of plant protein, but anti-nutritional factors such as condensed tannins reduce the biological value of their protein. Two recessive genes, zt-1 and zt-2, control the absence of tannins in faba bean seeds and also determine a white flower character on the plant. However, crosses between them produce coloured F₁ plants with tannins that contaminate the crop. Therefore, it is important to identify the gene present in all tannin-free cultivars and gene bank accessions to enable breeders to choose appropriate genitors for their crosses. The aim of this study was the identification of markers linked to zt-1, one of the genes governing free tannin content in faba bean. A segregating F₂ population derived from the cross between the coloured flower and high tannin content genotype Vf6 and a zt-1 line was developed and characterized phenotypically. Bulked Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to the zt-1 gene. Four RAPD loci (OPC5₅₅₁, OPG15₆₀₀, OPG11₁₁₇₁ and OPAF20₇₇₆) showed polymorphism between the contrasting bulks. The markers were sequenced to develop specific Sequence Characterised Amplified Regions (SCARs). Amplification of SCC5₅₅₁ produced a single product which was only observed in the white flowered and zero tannin content genotypes, whereas SCAR SCG11₁₁₇₁only produced a band in F₂ plants with coloured flower and high tannin content. SCARs SCC5₅₅₁ and SCG11₁₁₇₁ were tested for their applicability for routine screening in 37 faba bean genotypes differing in flower colour and tannin content. SCC5₅₅₁, allowed the prediction of the zt-1 genotypes with a 95% of accuracy, underscoring the potential of this SCAR marker as a cost-effective tool for MAS in large faba bean breeding populations.     

Protocol of a prospective study on the diagnostic value of transcranial duplex scanning of the substantia nigra in patients with parkinsonian symptoms

Background  

Parkinson's disease (PD) is the second most common neurodegenerative disorder. As there is no definitive diagnostic test, its diagnosis is based on clinical criteria. Recently transcranial duplex scanning (TCD) of the substantia nigra in the brainstem has been proposed as an instrument to diagnose PD. We and others have found that TCD scanning of substantia nigra duplex is a relatively accurate diagnostic instrument in patients with parkinsonian symptoms. However, all studies on TCD so far have involved well-defined, later-stage PD patients, which will obviously lead to an overestimate of the diagnostic accuracy of TCD.     

Tau regulates the localization and function of End‐binding proteins 1 and 3 in developing neuronal cells

The axonal microtubule‐associated protein tau is a well‐known regulator of microtubule stability in neurons. However, the putative interplay between tau and End‐binding proteins 1 and 3 (EB1/3), the core microtubule plus‐end tracking proteins, has not been elucidated yet. Here, we show that a cross‐talk between tau and EB1/3 exists in developing neuronal cells. Tau and EBs partially colocalize at extending neurites of N1E‐115 neuroblastoma cells and axons of primary hippocampal neurons, as shown by confocal immunofluorescence analyses. Tau down‐regulation leads to a reduction of EB1/3 comet length, as observed in shRNA‐stably depleted neuroblastoma cells and TAU?/? neurons. EB1/3 localization depends on the expression levels and localization of tau protein. Over‐expression of tau at high levels induces EBs relocalization to microtubule bundles at extending neurites of N1E‐115 cells. In differentiating primary neurons, tau is required for the proper accumulation of EBs at stretches of microtubule bundles at the medial and distal regions of the axon. Tau interacts with EB proteins, as shown by immunoprecipitation in different non‐neuronal and neuronal cells and in whole brain lysates. A tau/EB1 direct interaction was corroborated by in vitro pull‐down assays. Fluorescence recovery after photobleaching assays performed in neuroblastoma cells confirmed that tau modulates EB3 cellular mobility. In summary, we provide evidence of a new function of tau as a direct regulator of EB proteins in developing neuronal cells. This cross‐talk between a classical microtubule‐associated protein and a core microtubule plus‐end tracking protein may contribute to the fine‐tuned regulation of microtubule dynamics and stability during neuronal differentiation.

     

Model‐based approach to test hard polytomies in the Eulaemus clade of the most diverse South American lizard genus Liolaemus (Liolaemini,Squamata)

Lack of resolution in a phylogenetic tree is usually represented as a polytomy, and often adding more data (loci and taxa) resolves the species tree. These are the ‘soft’ polytomies, but in other cases additional data fail to resolve relationships; these are the ‘hard’ polytomies. This latter case is often interpreted as a simultaneous radiation of lineages in the history of a clade. Although hard polytomies are difficult to address, model‐based approaches provide new tools to test these hypotheses. Here, we used a clade of 144 species of the South American lizard clade Eulaemus to estimate phylogenies using a traditional concatenated matrix and three species tree methods: *BEAST, BEST, and minimizing deep coalescences (MDC). The different species tree methods recovered largely discordant results, but all resolved the same polytomy (e.g. very short internodes amongst lineages and low nodal support in Bayesian methods). We simulated data sets under eight explicit evolutionary models (including hard polytomies), tested these against empirical data (a total of 14 loci), and found support for two polytomies as the most plausible hypothesis for diversification of this clade. We discuss the performance of these methods and their limitations under the challenging scenario of hard polytomies. © 2015 The Linnean Society of London     

Ovariectomy differential influence on some hemostatic markers of mice and rats

Rodent ovariectomy is an experimental method to eliminate the main source of sexual steroids. This work explored for the first time the ovariectomy temporal changes induced in the hemostatic coagulation markers: prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration (FIB) along with uterine weight on adult female CD1 mice and Wistar rats. Uterine weight (Uw) was assessed before ovariectomy (control), and 1, 3, 5, 7, 9, 16, and 21 days after surgery. PT, aPTT, TT and FIB were estimated the same days, using reported standard techniques. Ovariectomy decreased Uw, since day 1; and from day 10 to 21 reached the lowest values for both species. After day 1, mice hemostatic parameters changed (PT +10%, P<0.05; aPTT +53%, P<0.05; TT −24%, P<0.05; FIB +67%, P<0.05). Rats showed significant changes only in TT and FIB (TT −13%, P<0.001; FIB +65%, P<0.001). Neither mice PT, aPTT and TT, recovered control values after 21 days. In the rats from day 5 to 16 aPTT diminished (18–23%, P<0.05) recovering to control values on day 21, TT after 9 days and PT on day 16. In both species, FIB returned to its control values after 9 days. Ovariectomy differentially altered the PT hemostatic parameter of mice and rats indicating a non-equivalence among both species behaviour for experimental studies of blood coagulation.     

<Emphasis Type="BoldItalic">In vivo</Emphasis> conditions influence the coding of stimulus features by bursts of action potentials

The functional role of burst firing (i.e. the firing of packets of action potentials followed by quiescence) in sensory processing is still under debate. Should bursts be considered as unitary events that signal the presence of a particular feature in the sensory environment or is information about stimulus attributes contained within their temporal structure? We compared the coding of stimulus attributes by bursts in vivo and in vitro of electrosensory pyramidal neurons in weakly electric fish by computing correlations between burst and stimulus attributes. Our results show that, while these correlations were strong in magnitude and significant in vitro, they were actually much weaker in magnitude if at all significant in vivo. We used a mathematical model of pyramidal neuron activity in vivo and showed that such a model could reproduce the correlations seen in vitro, thereby suggesting that differences in burst coding were not due to differences in bursting seen in vivo and in vitro. We next tested whether variability in the baseline (i.e. without stimulation) activity of ELL pyramidal neurons could account for these differences. To do so, we injected noise into our model whose intensity was calibrated to mimic baseline activity variability as quantified by the coefficient of variation. We found that this noise caused significant decreases in the magnitude of correlations between burst and stimulus attributes and could account for differences between in vitro and in vivo conditions. We then tested this prediction experimentally by directly injecting noise in vitro through the recording electrode. Our results show that this caused a lowering in magnitude of the correlations between burst and stimulus attributes in vitro and gave rise to values that were quantitatively similar to those seen under in vivo conditions. While it is expected that noise in the form of baseline activity variability will lower correlations between burst and stimulus attributes, our results show that such variability can account for differences seen in vivo. Thus, the high variability seen under in vivo conditions has profound consequences on the coding of information by bursts in ELL pyramidal neurons. In particular, our results support the viewpoint that bursts serve as a detector of particular stimulus features but do not carry detailed information about such features in their structure.     

Strategies for Neoglycan conjugation to human acid α-glucosidase

Engineering proteins for selective tissue targeting can improve therapeutic efficacy and reduce undesired side effects. The relatively high dose of recombinant human acid α-glucosidase (rhGAA) required for enzyme replacement therapy of Pompe disease may be attributed to less than optimal muscle uptake via the cation-independent mannose 6-phosphate receptor (CI-MPR). To improve muscle targeting, Zhu et al. (1) conjugated periodate oxidized rhGAA with bis mannose 6-phosphate bearing synthetic glycans and achieved 5-fold greater potency in a murine Pompe efficacy model. In the current study, we systematically evaluated multiple strategies for conjugation based on a structural homology model of GAA. Glycan derivatives containing succinimide, hydrazide, and aminooxy linkers targeting free cysteine, lysines, and N-linked glycosylation sites on rhGAA were prepared and evaluated in vitro and in vivo. A novel conjugation method using enzymatic oxidation was developed to eliminate side oxidation of methionine. Conjugates derived from periodate oxidized rhGAA still displayed the greatest potency in the murine Pompe model. The efficiency of conjugation and its effect on catalytic activity were consistent with predictions based on the structural model and supported its use in guiding selection of appropriate chemistries.     

Lizards from the end of the world: phylogenetic relationships of the Liolaemus lineomaculatus section (Squamata: Iguania: Liolaemini)

The Liolaemus lineomaculatus section is a geographically widely distributed group of lizards from the Patagonian region of southern South America, and includes 18 described species representing the most southerly distributed Liolaemus taxa (the genus includes 228 species and extends from Tierra del Fuego north to south-central Peru). Despite high species diversity, the phylogenetic relationships of this section are unknown. In the present work we sampled all described species in the L. lineomaculatus section as well as currently undescribed candidate species to reconstruct the first complete phylogenetic hypothesis for the clade. Our data set included four anonymous nuclear loci, three nuclear protein-coding loci, and two mitochondrial genes. We compared results obtained with three different phylogenetic methods for the concatenated data set (Maximum Parsimony, Maximum Likelihood and Bayesian Inference) with a coalescent-based species tree approach (BEST), and recovered congruent, strongly-supported topological arrangements across all methods. We identified four main clades within the L. lineomaculatus section: the lineomaculatus, magellanicus, somuncurae, and kingii+archeforus groups, for which we estimated divergence times. We discuss the taxonomic implications of these results and how the future integration of phylogeographic, niche modeling and morphological approaches will allow testing biogeographical hypotheses in this clade.