The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization,degradation, and toxicity

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson’s disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry–based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2_911–919, a nine amino acid segment within a flexible interdomain region (LRRK2_853–981), which we designate the “regulatory loop” (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2’s association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2’s interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase–dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.     

Regulation of nitrogen partitioning in field-grown almond trees: Effects of fruit load and foliar nitrogen applications

Two treatments were employed to influence the amount of amino nitrogen (N) transport in phloem. In walnut trees (Juglans regia L.), developing fruit significantly reduced the efflux of foliar-applied ¹⁵N-enriched urea from treated spurs over a 33-day period in comparison with similarly-treated defruited spurs. Those data suggest that local aboveground demand for N influences vascular transport of amino N. In another experiment, a 1% urea solution was applied foliarly to 5-year old `Mission' almond trees [Prunus dulcis (Mill.) D. A. Webb] to increase the concentration of amino N in the phloem. The effect of foliar N treatments on a) the transport and distribution of labelled urea N within the trees over the experimental period and b) the uptake of soil-applied labelled N were determined by replicated whole tree excavation, fractionation into various tree components and mass spectrometric analyses of the ¹⁴N/¹⁵N ratios. Concentrations and composition of amino acids in the phloem and xylem saps of control trees and trees receiving foliar-applied urea were also determined. In foliar urea-treated trees, the amino acid concentrations increased significantly in leaf and bark phloem exudate, within 24 and 96 h, respectively. Foliar-applied urea N was translocated to the roots of almond trees over the experimental period and decreased soil N uptake. The results of these experiments are consistent with the hypothesis that aboveground N demand affects the amount of amino N cycling between shoots and roots, and may be involved in the regulation of soil N uptake. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cutting the Gordian Knot: Phylogenetic and ecological diversification of the Mesalina brevirostris species complex (Squamata,Lacertidae)

Mesalina are small lacertid lizards occurring in the Saharo‐Sindian deserts from North Africa to the east of the Iranian plateau. Earlier phylogenetic studies indicated that there are several species complexes within the genus and that thorough taxonomic revisions are needed. In this study, we aim at resolving the phylogeny and taxonomy of the M. brevirostris species complex distributed from the Middle East to the Arabian/Persian Gulf region and Pakistan. We sequenced three mitochondrial and three nuclear gene fragments, and in combination with species delimitation and species‐tree estimation, we infer a time‐calibrated phylogeny of the complex. The results of the genetic analyses support the presence of four clearly delimited species in the complex that diverged approximately between the middle Pliocene and the Pliocene/Pleistocene boundary. Species distribution models of the four species show that the areas of suitable habitat are geographically well delineated and nearly allopatric, and that most of the species have rather divergent environmental niches. Morphological characters also confirm the differences between the species, although sometimes minute. As a result of all these lines of evidence, we revise the taxonomy of the Mesalina brevirostris species complex. We designate a lectotype for Mesalina brevirostris Blanford, 1874; resurrect the available name Eremias bernoullii Schenkel, 1901 from the synonymy of M. brevirostris; elevate M. brevirostris microlepis (Angel, 1936) to species status; and describe Mesalina saudiarabica, a new species from Saudi Arabia.     

Thermodynamics of Aβ16–21 dissociation from a fibril: Enthalpy,entropy, and volumetric properties

Here, we provide insights into the thermodynamic properties of A dissociation from an amyloid fibril using all‐atom molecular dynamics simulations in explicit water. An umbrella sampling protocol is used to compute potentials of mean force (PMF) as a function of the distance ξ between centers‐of‐mass of the A peptide and the preformed fibril at nine temperatures. Changes in the enthalpy and the entropic energy are determined from the temperature dependence of these PMF(s) and the average volume of the simulation box is computed as a function of ξ. We find that the PMF at 310 K is dominated by enthalpy while the entropic energy does not change significantly during dissociation. The volume of the system decreases during dissociation. Moreover, the magnitude of this volume change also decreases with increasing temperature. By defining dock and lock states using the solvent accessible surface area (SASA), we find that the behavior of the electrostatic energy is different in these two states. It increases (unfavorable) and decreases (favorable) during dissociation in lock and dock states, respectively, while the energy due to Lennard‐Jones interactions increases continuously in these states. Our simulations also highlight the importance of hydrophobic interactions in accounting for the stability of A . Proteins 2015; 83:1963–1972. © 2015 Wiley Periodicals, Inc.     

Fibrous Hydrogels for Cell Encapsulation: A Modular and Supramolecular Approach

Artificial 3-dimensional (3D) cell culture systems, which mimic the extracellular matrix (ECM), hold great potential as models to study cellular processes under controlled conditions. The natural ECM is a 3D structure composed of a fibrous hydrogel that provides both mechanical and biochemical cues to instruct cell behavior. Here we present an ECM-mimicking genetically engineered protein-based hydrogel as a 3D cell culture system that combines several key features: (1) Mild and straightforward encapsulation meters (1) ease of ut I am not so sure.encapsulation of the cells, without the need of an external crosslinker. (2) Supramolecular assembly resulting in a fibrous architecture that recapitulates some of the unique mechanical characteristics of the ECM, i.e. strain-stiffening and self-healing behavior. (3) A modular approach allowing controlled incorporation of the biochemical cue density (integrin binding RGD domains). We tested the gels by encapsulating MG-63 osteoblastic cells and found that encapsulated cells not only respond to higher RGD density, but also to overall gel concentration. Cells in 1% and 2% (weight fraction) protein gels showed spreading and proliferation, provided a relative RGD density of at least 50%. In contrast, in 4% gels very little spreading and proliferation occurred, even for a relative RGD density of 100%. The independent control over both mechanical and biochemical cues obtained in this modular approach renders our hydrogels suitable to study cellular responses under highly defined conditions.     

Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells

Vascular endothelial growth factor (VEGF) is an essential regulator of normal and abnormal blood vessel growth. A monoclonal antibody (mAb) that targets VEGF suppresses tumor growth in murine cancer models and human patients. We investigated cellular and molecular events that mediate refractoriness of tumors to anti-angiogenic therapy. Inherent anti-VEGF refractoriness is associated with infiltration of the tumor tissue by CD11b+Gr1+ myeloid cells. Recruitment of these myeloid cells is also sufficient to confer refractoriness. Combining anti-VEGF treatment with a mAb that targets myeloid cells inhibits growth of refractory tumors more effectively than anti-VEGF alone. Gene expression analysis in CD11b+Gr1+ cells isolated from the bone marrow of mice bearing refractory tumors reveals higher expression of a distinct set of genes known to be implicated in active mobilization and recruitment of myeloid cells. These findings indicate that, in our models, refractoriness to anti-VEGF treatment is determined by the ability of tumors to prime and recruit CD11b+Gr1+ cells.     

Cooperation of sonic hedgehog enhancers in midline expression

In zebrafish, as in other vertebrates, the secreted signalling molecule Sonic hedgehog (Shh) is expressed in organiser regions such as the embryonic midline and the zona limitans intrathalamica (zli). To investigate the regulatory mechanisms underlying the pattern of shh expression, we carried out a systematic analysis of the intronic regulatory sequences of zebrafish shh using stable transgenesis. Deletion analysis identified the modules responsible for expression in the embryonic shield, the hypothalamus and the zli and confirmed the activities of previously identified notochord and floor plate enhancers. We detected a strong synergism between regulatory regions. The degree of synergy varied over time in the hypothalamus suggesting different mechanisms for initiation and maintenance of expression. Our data show that the pattern of shh expression in the embryonic central nervous system involves an intricate crosstalk of at least 4 different regulatory regions. When compared to the enhancer activities of the mouse Shh gene, we observed a remarkable divergence of function of structurally conserved enhancer sequences. The activating region ar-C (61% identical to SFPE2 in mouse Shh), for example, mediates floor plate expression in the mouse embryo while it directs expression in the forebrain and the notochord and only weakly in the floor plate in the zebrafish embryo. This raises doubts on the predictive power of phylogenetic footprinting and indicates a stunning divergence of function of structurally conserved regulatory modules during vertebrate evolution.     

Molecular phylogeny of selected Old World Astragalus (Fabaceae): incongruence among chloroplast trnL-F, ndhF and nuclear ribosomal DNA ITS sequences

This study reports maximum parsimony and Bayesian phylogenetic analyses of selected Old World Astragalus using two chloroplast fragments including trnL-F and ndhF and the nuclear ribosomal internal transcribed spacer (nrDNA ITS). A total of 52 taxa including 34 euploid Old World and New World Astragalus , one aneuploid species from the Neo-Astragalus clade as a representative and 14 other Astragalean taxa, plus Cheseneya astragalina and two species of Caragana as outgroups were analyzed for both trnL-F and nrDNA ITS regions. ndhF was analyzed in 30 taxa and the same number for the combination of these three datasets were examined. In general, the trnL-F dataset and the ndhF and nrDNA ITS datasets generated more or less the same clades within Astragalus . However, in the trnL-F and ndhF phylogenies, Astragalus species are not gathered in a single clade, the so-called Astragalus s.s., as indicated by the nrDNA ITS tree. Visual inspection of these three phylogenies revealed that they were inconsistent regarding the position and relationships of Astragalus hemsleyi , A. ophiocarpus , A. annularis–A. epiglottis / Astragalus pelecinus, A. echinatus and A. arizonicus . Incongruence length difference test suggested that the trnL-F , ndhF and nrDNA ITS datasets were incongruent. In spite of this, phylogenetic analyses of the combined datasets as one unit or as three partitions generated trees that were topologically similar as a mix of the cpDNA and the nrDNA ITS trees. However, the combined dataset provided more resolved and statistically supported clades. The recently described A. memoriosus appeared closely related to A. stocksii (both from sect. Caraganella ) based on both trnL-F and nrDNA ITS sequences.     

MECP2 Isoform-Specific Vectors with Regulated Expression for Rett Syndrome Gene Therapy

Background

Rett Syndrome (RTT) is an Autism Spectrum Disorder and the leading cause of mental retardation in females. RTT is caused by mutations in the Methyl CpG-Binding Protein-2 (MECP2) gene and has no treatment. Our objective is to develop viral vectors for MECP2 gene transfer into Neural Stem Cells (NSC) and neurons suitable for gene therapy of Rett Syndrome.

Methodology/Principal Findings

We generated self-inactivating (SIN) retroviral vectors with the ubiquitous EF1α promoter avoiding known silencer elements to escape stem-cell-specific viral silencing. High efficiency NSC infection resulted in long-term EGFP expression in transduced NSC and after differentiation into neurons. Infection with Myc-tagged MECP2-isoform-specific (E1 and E2) vectors directed MeCP2 to heterochromatin of transduced NSC and neurons. In contrast, vectors with an internal mouse Mecp2 promoter (MeP) directed restricted expression only in neurons and glia and not NSC, recapitulating the endogenous expression pattern required to avoid detrimental consequences of MECP2 ectopic expression. In differentiated NSC from adult heterozygous Mecp2^tm1.1Bird+/− female mice, 48% of neurons expressed endogenous MeCP2 due to random inactivation of the X-linked Mecp2 gene. Retroviral MECP2 transduction with EF1α and MeP vectors rescued expression in 95–100% of neurons resulting in increased dendrite branching function in vitro. Insulated MECP2 isoform-specific lentiviral vectors show long-term expression in NSC and their differentiated neuronal progeny, and directly infect dissociated murine cortical neurons with high efficiency.

Conclusions/Significance

MeP vectors recapitulate the endogenous expression pattern of MeCP2 in neurons and glia. They have utility to study MeCP2 isoform-specific functions in vitro, and are effective gene therapy vectors for rescuing dendritic maturation of neurons in an ex vivo model of RTT.