Teaching old NCATs new tricks: using non-canonical amino acid tagging to study neuronal plasticity

The non-canonical amino acid labeling techniques BONCAT (bioorthogonal non-canonical amino acid tagging) and FUNCAT (fluorescent non-canonical amino acid tagging) enable the specific identification and visualization of newly synthesized proteins. Recently, these techniques have been applied to neuronal systems to elucidate protein synthesis dynamics during plasticity, identify stimulation-induced proteomes and subproteomes and to investigate local protein synthesis in specific subcellular compartments. The next generation of tools and applications, reviewed here, includes the development of new tags, the quantitative identification of newly synthesized proteins, the application of NCAT to whole animals, and the ability to genetically restrict NCAT labeling. These techniques will enable not only improved detection but also allow new scientific questions to be tackled

Charge-density Waves Survive the Pauli Paramagnetic Limit

Measurements of the resistance of single crystals of (Per)$_2$Au(mnt)$_2$ have been made at magnetic fields $B$ of up to 45 T, exceeding the Pauli paramagnetic limit of $B_{\rm P}\approx 37$ T. The continued presence of non-linear charge-density wave electrodynamics at $B \geq 37$ T unambiguously establishes the survival of the charge-density wave state above the Pauli paramagnetic limit, and the likely emergence of an inhomogeneous phase analogous to that anticipated to occur in superconductors.Comment: 4 pages, three figure

Scope and significance of non-uniform classification practices in breast cancer with non-inflammatory skin involvement: a clinicopathologic study and an international survey

Background: The study evaluates the scope of non-uniform classification practices concerning breast carcinomas with non-inflammatory skin involvement. Patients and methods: We compared the clinical course of patients with histologically proven non-inflammatory skin involvement: 119 (65.4%) with clinically obvious ‘classical' skin changes (Group A) and 63 (34.6%) with no or only discreet changes (Group B). A questionnaire was circulated to pathology departments in 24 countries to assess the practice concerning the placement of skin- involved breast carcinomas in the TNM classification. Results: Patients in Group B showed a significantly better disease specific survival (P = 0.0002). Eighty-six respondents (70.5%) of the survey preferred the ‘histological view' and classified tumors with only histological proven skin involvement as T4b/stage IIIB. The opposing classification principle (‘clinical view'), which dictates that T4b breast cancer is a clinical diagnosis and the classical signs must be present, was supported by 31 respondents (25.4%). Conclusions: A large number of breast cancer patients with non-inflammatory skin involvement are only histologically proven and show, compared with cases exhibiting the classical clinical signs, significant differences in clinical course and prognosis. In general, both subsets were aggregated in one T category/stage (T4b/IIIB). This results in a considerable distortion of the reported statistical dat

High magnetic field induced charge density wave states in a quasi-one dimensional organic conductor

We have measured the high field magnetoresistence and magnetization of quasi-one- dimensional (Q1D) organic conductor (Per)2Pt(mnt)2 (where Per = perylene and mnt = maleonitriledithiolate), which has a charge density wave (CDW) ground state at zero magnetic field below 8 K. We find that the CDW ground state is suppressed with moderate magnetic fields of order 20 T, as expected from a mean field theory treatment of Pauli effects[W. Dieterich and P. Fulde, Z. Physik 265, 239 - 243 (1973)]. At higher magnetic fields, a new, density wave state with sub-phases is observed in the range 20 to 50 T, which is reminiscent of the cascade of field induced, quantized, spin density wave phases (FISDW) observed in the Bechgaard salts. The new density wave state, which we tenatively identify as a field induced charge density wave state (FICDW), is re-entrant to a low resistance state at even higher fields, of order 50 T and above. Unlike the FISDW ground state, the FICDW state is only weakly orbital, and appears for all directions of magnetic field. Our findings are substantiated by electrical resistivity, magnetization, thermoelectric, and Hall measurements. We discuss our results in light of theoretical work involving magnetic field dependent Q1D CDW ground states in high magnetic fields [D. Zanchi, A. Bjelis, and G. Montambaux, Phys. Rev. B 53, (1996)1240; A. Lebed, JETP Lett. 78,138(2003)].Comment: 16 pages, 5 figure

Deregulated splicing is a major mechanism of RNA-induced toxicity in Huntington's disease

Huntington's disease (HD) is caused by an expanded CAG repeat in the huntingtin (HTT) gene, translating into an elongated polyglutamine stretch. In addition to the neurotoxic mutant HTT protein, the mutant CAG repeat RNA can exert toxic functions by trapping RNA-binding proteins. While few examples of proteins that aberrantly bind to mutant HTT RNA and execute abnormal function in conjunction with the CAG repeat RNA have been described, an unbiased approach to identify the interactome of mutant HTT RNA is missing. Here, we describe the analysis of proteins that preferentially bind mutant HTT RNA using a mass spectrometry approach. We show that (I) the majority of proteins captured by mutant HTT RNA belong to the spliceosome pathway, (II) expression of mutant CAG repeat RNA induces mis-splicing in a HD cell model, (III) overexpression of one of the splice factors trapped by mutant HTT ameliorates the HD phenotype in a fly model and (VI) deregulated splicing occurs in human HD brain. Our data suggest that deregulated splicing is a prominent mechanism of RNA-induced toxicity in HD

Simulational study of anomalous tracer diffusion in hydrogels

In this article, we analyze different factors that affect the diffusion behavior of small tracer particles (as they are used e.g.in fluorescence correlation spectroscopy (FCS)) in the polymer network of a hydrogel and perform simulations of various simplified models. We observe, that under certain circumstances the attraction of a tracer particle to the polymer network strands might cause subdiffusive behavior on intermediate time scales. In theory, this behavior could be employed to examine the network structure and swelling behavior of weakly crosslinked hydrogels with the help of FCS.Comment: 11 pages, 11 figure

Noncanonical Amino Acid Labeling in Vivo to Visualize and Affinity Purify Newly Synthesized Proteins in Larval Zebrafish

Protein expression in the nervous system undergoes regulated changes in response to changes in behavioral states, in particular long-term memory formation. Recently, methods have been developed (BONCAT and FUNCAT), which introduce noncanonical amino acids bearing small bio-orthogonal functional groups into proteins using the cells’ own translational machinery. Using the selective “click reaction”, this allows for the identification and visualization of newly synthesized proteins in vitro. Here we demonstrate that noncanonical amino acid labeling can be achieved in vivo in an intact organism capable of simple learning behavior, the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, stimulation with a GABA antagonist that elicits seizures in the larval zebrafish causes an increase in protein synthesis throughout the proteome, which can also be visualized in intact larvae

Cell-selective metabolic labeling of proteins

Metabolic labeling of proteins with the methionine surrogate azidonorleucine can be targeted exclusively to specified cells through expression of a mutant methionyl-tRNA synthetase (MetRS). In complex cellular mixtures, proteins made in cells that express the mutant synthetase can be tagged with affinity reagents (for detection or enrichment) or fluorescent dyes (for imaging). Proteins made in cells that do not express the mutant synthetase are neither labeled nor detected

Cortical Factor Feedback Model for Cellular Locomotion and Cytofission

Eukaryotic cells can move spontaneously without being guided by external cues. For such spontaneous movements, a variety of different modes have been observed, including the amoeboid-like locomotion with protrusion of multiple pseudopods, the keratocyte-like locomotion with a widely spread lamellipodium, cell division with two daughter cells crawling in opposite directions, and fragmentations of a cell to multiple pieces. Mutagenesis studies have revealed that cells exhibit these modes depending on which genes are deficient, suggesting that seemingly different modes are the manifestation of a common mechanism to regulate cell motion. In this paper, we propose a hypothesis that the positive feedback mechanism working through the inhomogeneous distribution of regulatory proteins underlies this variety of cell locomotion and cytofission. In this hypothesis, a set of regulatory proteins, which we call cortical factors, suppress actin polymerization. These suppressing factors are diluted at the extending front and accumulated at the retracting rear of cell, which establishes a cellular polarity and enhances the cell motility, leading to the further accumulation of cortical factors at the rear. Stochastic simulation of cell movement shows that the positive feedback mechanism of cortical factors stabilizes or destabilizes modes of movement and determines the cell migration pattern. The model predicts that the pattern is selected by changing the rate of formation of the actin-filament network or the threshold to initiate the network formation

Polarization transfer in the 4^{4}He(e⃗,e′p⃗3(\vec{e},e' \vec{p}^{3}H reaction

Polarization transfer in the 4He(e,e'p)3H reaction at a Q^2 of 0.4 (GeV/c)^2 was measured at the Mainz Microtron MAMI. The ratio of the transverse to the longitudinal polarization components of the ejected protons was compared with the same ratio for elastic ep scattering. The results are consistent with a recent fully relativistic calculation which includes a predicted medium modification of the proton form factor based on a quark-meson coupling model.Comment: 5 pages, Latex, 2 postscript figures, submitted to Physics Letters