The Development of Cell Processes Induced by tau Protein Requires Phosphorylation of Serine 262 and 356 in the Repeat Domain and Is Inhibited by Phosphorylation in the Proline-rich Domains

The differentiation of neurons and the outgrowth of neurites depends on microtubule-associated proteins such as tau protein. To study this process, we have used the model of Sf9 cells, which allows efficient transfection with microtubule-associated proteins (via baculovirus vectors) and observation of the resulting neurite-like extensions. We compared the phosphorylation of tau23 (the embryonic form of human tau) with mutants in which critical phosphorylation sites were deleted by mutating Ser or Thr residues into Ala. One can broadly distinguish two types of sites, the KXGS motifs in the repeats (which regulate the affinity of tau to microtubules) and the SP or TP motifs in the domains flanking the repeats (which contain epitopes for antibodies diagnostic of Alzheimer’s disease). Here we report that both types of sites can be phosphorylated by endogenous kinases of Sf9 cells, and that the phosphorylation pattern of the transfected tau is very similar to that of neurons, showing that Sf9 cells can be regarded as an approximate model for the neuronal balance between kinases and phosphatases. We show that mutations in the repeat domain and in the flanking domains have opposite effects. Mutations of KXGS motifs in the repeats (Ser262, 324, and 356) strongly inhibit the outgrowth of cell extensions induced by tau, even though this type of phosphorylation accounts for only a minor fraction of the total phosphate. This argues that the temporary detachment of tau from microtubules (by phosphorylation at KXGS motifs) is a necessary condition for establishing cell polarity at a critical point in space or time. Conversely, the phosphorylation at SP or TP motifs represents the majority of phosphate (>80%); mutations in these motifs cause an increase in cell extensions, indicating that this type of phosphorylation retards the differentiation of the cells.

Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues

p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer–dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.

Functional cell surface expression of the anion transport domain of human red cell band 3 (AE1) in the yeast Saccharomyces cerevisiae.

We expressed the 52-kDa integral membrane domain (B3mem) of the human erythrocyte anion transporter (band 3; AE1) in a protease-deficient strain of the yeast Saccharomyces cerevisiae under the control of the inducible GAL10-CYC1 promoter. Immunoblots of total protein from transformed yeast cells confirmed that the B3mem polypeptide was overexpressed shortly after induction with galactose. Cell surface expression of the functional anion transporter was detected by using a simple transport assay to measure stilbene disulfonate-inhibitable chloride influx into intact yeast cells. The B3mem polypeptide was recycled and degraded by the cells with a half-life of approximately 1-3 hr, which led to a steady-state level of expression in exponentially growing cultures. Our data suggest that 5-10% of total B3mem is functionally active at the cell surface at any one time and that overexpression of this anion transport protein does not interfere with cell growth or survival. This is one of only a few reports of the functional expression of a plasma membrane transport protein in the plasma membrane of yeast cells and to our knowledge is the first report of red cell band 3-mediated anion transport at the plasma membrane of cDNA-transformed cells. The cell surface expression system we describe will provide a simple means for future study of the functional properties of band 3 by using site-directed mutagenesis.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules.

The WW domain has previously been described as a motif of 38 semiconserved residues found in seemingly unrelated proteins, such as dystrophin, Yes-associated protein (YAP), and two transcriptional regulators, Rsp-5 and FE65. The molecular function of the WW domain has been unknown until this time. Using a functional screen of a cDNA expression library, we have identified two putative ligands of the WW domain of YAP, which we named WBP-1 and WBP-2. Peptide sequence comparison between the two partial clones revealed a homologous region consisting of a proline-rich domain followed by a tyrosine residue (with the shared sequence PPPPY), which we shall call the PY motif. Binding assays and site-specific mutagenesis have shown that the PY motif binds with relatively high affinity and specificity to the WW domain of YAP, with the preliminary consensus XPPXY being critical for binding. Herein, we have implicated the WW domain with a role in mediating protein-protein interactions, as a variant of the paradigm set by Src homology 3 domains and their proline-rich ligands.

Sentiment classification for early detection of health alerts in the chemical textile domain

In the chemical textile domain experts have to analyse chemical components and substances that might be harmful for their usage in clothing and textiles. Part of this analysis is performed searching opinions and reports people have expressed concerning these products in the Social Web. However, this type of information on the Internet is not as frequent for this domain as for others, so its detection and classification is difficult and time-consuming. Consequently, problems associated to the use of chemical substances in textiles may not be detected early enough, and could lead to health problems, such as allergies or burns. In this paper, we propose a framework able to detect, retrieve, and classify subjective sentences related to the chemical textile domain, that could be integrated into a wider health surveillance system. We also describe the creation of several datasets with opinions from this domain, the experiments performed using machine learning techniques and different lexical resources such as WordNet, and the evaluation focusing on the sentiment classification, and complaint detection (i.e., negativity). Despite the challenges involved in this domain, our approach obtains promising results with an F-score of 65% for polarity classification and 82% for complaint detection.

TrendMiner: Large-scale Cross-lingual Trend Mining Summarization of Real-time Media Streams

El reciente crecimiento masivo de medios on-line y el incremento de los contenidos generados por los usuarios (por ejemplo, weblogs, Twitter, Facebook) plantea retos en el acceso e interpretación de datos multilingües de manera eficiente, rápida y asequible. El objetivo del proyecto TredMiner es desarrollar métodos innovadores, portables, de código abierto y que funcionen en tiempo real para generación de resúmenes y minería cross-lingüe de medios sociales a gran escala. Los resultados se están validando en tres casos de uso: soporte a la decisión en el dominio financiero (con analistas, empresarios, reguladores y economistas), monitorización y análisis político (con periodistas, economistas y políticos) y monitorización de medios sociales sobre salud con el fin de detectar información sobre efectos adversos a medicamentos.

Dynamical Approach for Real-Time Monitoring of Agricultural Crops

In this paper, a novel approach for exploiting multitemporal remote sensing data focused on real-time monitoring of agricultural crops is presented. The methodology is defined in a dynamical system context using state-space techniques, which enables the possibility of merging past temporal information with an update for each new acquisition. The dynamic system context allows us to exploit classical tools in this domain to perform the estimation of relevant variables. A general methodology is proposed, and a particular instance is defined in this study based on polarimetric radar data to track the phenological stages of a set of crops. A model generation from empirical data through principal component analysis is presented, and an extended Kalman filter is adapted to perform phenological stage estimation. Results employing quad-pol Radarsat-2 data over three different cereals are analyzed. The potential of this methodology to retrieve vegetation variables in real time is shown.

GTInspector: A Moldable Domain-Aware Object Inspector

Understanding the run-time behaviour of object-oriented applications entails the comprehension of run-time objects. Traditional object inspectors favor generic views that focus on the low-level details of the state of single objects. While universally applicable, this generic approach does not take into account the varying needs of developers that could benefit from tailored views and exploration possibilities. GTInspector is a novel moldable object inspector that provides different high-level ways to visualize and explore objects, adapted to both the object and the current developer need.

Practical domain-specific debuggers using the Moldable Debugger framework

Understanding the run-time behavior of software systems can be a challenging activity. Debuggers are an essential category of tools used for this purpose as they give developers direct access to the running systems. Nevertheless, traditional debuggers rely on generic mechanisms to introspect and interact with the running systems, while developers reason about and formulate domain-specific questions using concepts and abstractions from their application domains. This mismatch creates an abstraction gap between the debugging needs and the debugging support leading to an inefficient and error-prone debugging effort, as developers need to recover concrete domain concepts using generic mechanisms. To reduce this gap, and increase the efficiency of the debugging process, we propose a framework for developing domain-specific debuggers, called the Moldable Debugger, that enables debugging at the level of the application domain. The Moldable Debugger is adapted to a domain by creating and combining domain-specific debugging operations with domain-specific debugging views, and adapts itself to a domain by selecting, at run time, appropriate debugging operations and views. To ensure the proposed model has practical applicability (i.e., can be used in practice to build real debuggers), we discuss, from both a performance and usability point of view, three implementation strategies. We further motivate the need for domain-specific debugging, identify a set of key requirements and show how our approach improves debugging by adapting the debugger to several domains.

Gradient-Domain Bidirectional Path Tracing

Gradient-domain path tracing has recently been introduced as an efficient realistic image synthesis algorithm. This paper introduces a bidirectional gradient-domain sampler that outperforms traditional bidirectional path tracing often by a factor of two to five in terms of squared error at equal render time. It also improves over unidirectional gradient-domain path tracing in challenging visibility conditions, similarly as conventional bidirectional path tracing improves over its unidirectional counterpart. Our algorithm leverages a novel multiple importance sampling technique and an efficient implementation of a high-quality shift mapping suitable for bidirectional path tracing. We demonstrate the versatility of our approach in several challenging light transport scenarios.

The story of the diamond necklace, told in detail for the first time, by the aid of contemporary memoires, original letters, and official and other documents; and comprising a sketch of the life of the Countess de la Motte, pretended confidant of Marie-Antoinette, with particulars of the careers of the other actors in the remarkable drama.

"Authorities referred to in this work": p.ix-xii.

Time-critical aid : questionable use of disaster assistance funds for Peru : report to the Administrator, Agency for International Development /

"B-220921"--P. 1.

Essays and addresses in war time.

Mode of access: Internet.

Automatic safety belt systems: changes in owner usage over time in GM Chevettes and VW Rabbits. Final report.

National Highway Traffic Safety Administration, Washington, D.C.