Free energy of burying hydrophobic residues in the interface between protein subunits

We have obtained an experimental estimate of the free energy change associated with variations at the interface between protein subunits, a subject that has raised considerable interest since the concept of accessible surface area was introduced by Lee and Richards [Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379–400]. We determined by analytical ultracentrifugation the dimer–tetramer equilibrium constant of five single and three double mutants of human Hb. One mutation is at the stationary α1β1 interface, and all of the others are at the sliding α1β2 interface where cleavage of the tetramer into dimers and ligand-linked allosteric changes are known to occur. A surprisingly good linear correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area was obtained, after corrections for the energetic cost of losing steric complementarity at the αβ dimer interface. The slope yields an interface stabilization free energy of −15 ± 1.2 cal/mol upon burial of 1 Å2 of hydrophobic surface, in very good agreement with the theoretical estimate given by Eisenberg and McLachlan [Eisenberg, D. & McLachlan, A. D. (1986) Nature (London) 319, 199–203].

Engineering actin-resistant human DNase I for treatment of cystic fibrosis.

Human deoxyribonuclease I (DNase I), an enzyme recently approved for treatment of cystic fibrosis (CF), has been engineered to create two classes of mutants: actin-resistant variants, which still catalyze DNA hydrolysis but are no longer inhibited by globular actin (G-actin) and active site variants, which no longer catalyze DNA hydrolysis but still bind G-actin. Actin-resistant variants with the least affinity for actin, as measured by an actin binding ELISA and actin inhibition of [33P] DNA hydrolysis, resulted from the introduction of charged, aliphatic, or aromatic residues at Ala-114 or charged residues on the central hydrophobic actin binding interface at Tyr-65 or Val-67. In CF sputum, the actin-resistant variants D53R, Y65A, Y65R, or V67K were 10-to 50-fold more potent than wild type in reducing viscoelasticity as determined in sputum compaction assays. The reduced viscoelasticity correlated with reduced DNA length as measured by pulsed-field gel electrophoresis. In contrast, the active site variants H252A or H134A had no effect on altering either viscoelasticity or DNA length in CF sputum. The data from both the active site and actin-resistant variants demonstrate that the reduction of viscoelasticity by DNase I results from DNA hydrolysis and not from depolymerization of filamentous actin (F-actin). The increased potency of the actin-resistant variants indicates that G-actin is a significant inhibitor of DNase I in CF sputum. These results further suggest that actin-resistant DNase I variants may have improved efficacy in CF patients.

Crystal structure at 2.6-A resolution of human macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) was the first cytokine to be described, but for 30 years its role in the immune response remained enigmatic. In recent studies, MIF has been found to be a novel pituitary hormone and the first protein identified to be released from immune cells on glucocorticoid stimulation. Once secreted, MIF counterregulates the immunosuppressive effects of steroids and thus acts as a critical component of the immune system to control both local and systemic immune responses. We report herein the x-ray crystal structure of human MIF to 2.6 angstrom resolution. The protein is a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has an additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three beta-sheets are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular 3-fold axis. Electrostatic potential maps reveal that the channel has a positive potential, suggesting that it binds negatively charged molecules. The elucidated structure for MIF is unique among cytokines or hormonal mediators, and suggests that this counterregulator of glucocorticoid action participates in novel ligand-receptor interactions.

Human cooperation in the simultaneous and the alternating Prisoner's Dilemma: Pavlov versus Generous Tit-for-Tat.

The iterated Prisoner's Dilemma has become the paradigm for the evolution of cooperation among egoists. Since Axelrod's classic computer tournaments and Nowak and Sigmund's extensive simulations of evolution, we know that natural selection can favor cooperative strategies in the Prisoner's Dilemma. According to recent developments of theory the last champion strategy of "win--stay, lose--shift" ("Pavlov") is the winner only if the players act simultaneously. In the more natural situation of players alternating the roles of donor and recipient a strategy of "Generous Tit-for-Tat" wins computer simulations of short-term memory strategies. We show here by experiments with humans that cooperation dominated in both the simultaneous and the alternating Prisoner's Dilemma. Subjects were consistent in their strategies: 30% adopted a Generous Tit-for-Tat-like strategy, whereas 70% used a Pavlovian strategy in both the alternating and the simultaneous game. As predicted for unconditional strategies, Pavlovian players appeared to be more successful in the simultaneous game whereas Generous Tit-for-Tat-like players achieved higher payoffs in the alternating game. However, the Pavlovian players were smarter than predicted: they suffered less from defectors and exploited cooperators more readily. Humans appear to cooperate either with a Generous Tit-for-Tat-like strategy or with a strategy that appreciates Pavlov's advantages but minimizes its handicaps.

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition.

Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor alpha chain (hIL-5R alpha). A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor alpha chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5R alpha were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues Glu-110 and Trp-111, and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important alpha chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5R alpha.

Scientific bases of human-machine communication by voice.

The scientific bases for human-machine communication by voice are in the fields of psychology, linguistics, acoustics, signal processing, computer science, and integrated circuit technology. The purpose of this paper is to highlight the basic scientific and technological issues in human-machine communication by voice and to point out areas of future research opportunity. The discussion is organized around the following major issues in implementing human-machine voice communication systems: (i) hardware/software implementation of the system, (ii) speech synthesis for voice output, (iii) speech recognition and understanding for voice input, and (iv) usability factors related to how humans interact with machines.

Commercial applications of speech interface technology: an industry at the threshold.

Speech interface technology, which includes automatic speech recognition, synthetic speech, and natural language processing, is beginning to have a significant impact on business and personal computer use. Today, powerful and inexpensive microprocessors and improved algorithms are driving commercial applications in computer command, consumer, data entry, speech-to-text, telephone, and voice verification. Robust speaker-independent recognition systems for command and navigation in personal computers are now available; telephone-based transaction and database inquiry systems using both speech synthesis and recognition are coming into use. Large-vocabulary speech interface systems for document creation and read-aloud proofing are expanding beyond niche markets. Today's applications represent a small preview of a rich future for speech interface technology that will eventually replace keyboards with microphones and loud-speakers to give easy accessibility to increasingly intelligent machines.

Military and government applications of human-machine communication by voice.

This paper describes a range of opportunities for military and government applications of human-machine communication by voice, based on visits and contacts with numerous user organizations in the United States. The applications include some that appear to be feasible by careful integration of current state-of-the-art technology and others that will require a varying mix of advances in speech technology and in integration of the technology into applications environments. Applications that are described include (1) speech recognition and synthesis for mobile command and control; (2) speech processing for a portable multifunction soldier's computer; (3) speech- and language-based technology for naval combat team tactical training; (4) speech technology for command and control on a carrier flight deck; (5) control of auxiliary systems, and alert and warning generation, in fighter aircraft and helicopters; and (6) voice check-in, report entry, and communication for law enforcement agents or special forces. A phased approach for transfer of the technology into applications is advocated, where integration of applications systems is pursued in parallel with advanced research to meet future needs.

3D visual sensing of human hand for remote operation of a robotic hand

New low cost sensors and open free libraries for 3D image processing are making important advances in robot vision applications possible, such as three-dimensional object recognition, semantic mapping, navigation and localization of robots, human detection and/or gesture recognition for human-machine interaction. In this paper, a novel method for recognizing and tracking the fingers of a human hand is presented. This method is based on point clouds from range images captured by a RGBD sensor. It works in real time and it does not require visual marks, camera calibration or previous knowledge of the environment. Moreover, it works successfully even when multiple objects appear in the scene or when the ambient light is changed. Furthermore, this method was designed to develop a human interface to control domestic or industrial devices, remotely. In this paper, the method was tested by operating a robotic hand. Firstly, the human hand was recognized and the fingers were detected. Secondly, the movement of the fingers was analysed and mapped to be imitated by a robotic hand.

How to make a natural language interface to query databases accessible to everyone: An example

Natural Language Interfaces to Query Databases (NLIDBs) have been an active research field since the 1960s. However, they have not been widely adopted. This article explores some of the biggest challenges and approaches for building NLIDBs and proposes techniques to reduce implementation and adoption costs. The article describes {AskMe*}, a new system that leverages some of these approaches and adds an innovative feature: query-authoring services, which lower the entry barrier for end users. Advantages of these approaches are proven with experimentation. Results confirm that, even when {AskMe*} is automatically reconfigurable against multiple domains, its accuracy is comparable to domain-specific NLIDBs.

RGBD Human-Hand recognition for the Interaction with Robot-Hand

New low cost sensors and the new open free libraries for 3D image processing are permitting to achieve important advances for robot vision applications such as tridimensional object recognition, semantic mapping, navigation and localization of robots, human detection and/or gesture recognition for human-machine interaction. In this paper, a method to recognize the human hand and to track the fingers is proposed. This new method is based on point clouds from range images, RGBD. It does not require visual marks, camera calibration, environment knowledge and complex expensive acquisition systems. Furthermore, this method has been implemented to create a human interface in order to move a robot hand. The human hand is recognized and the movement of the fingers is analyzed. Afterwards, it is imitated from a Barret hand, using communication events programmed from ROS.

Experiences Using an Open Source Sofware Library to Teach Computer Vision Subjects

Machine vision is an important subject in computer science and engineering degrees. For laboratory experimentation, it is desirable to have a complete and easy-to-use tool. In this work we present a Java library, oriented to teaching computer vision. We have designed and built the library from the scratch with enfasis on readability and understanding rather than on efficiency. However, the library can also be used for research purposes. JavaVis is an open source Java library, oriented to the teaching of Computer Vision. It consists of a framework with several features that meet its demands. It has been designed to be easy to use: the user does not have to deal with internal structures or graphical interface, and should the student need to add a new algorithm it can be done simply enough. Once we sketch the library, we focus on the experience the student gets using this library in several computer vision courses. Our main goal is to find out whether the students understand what they are doing, that is, find out how much the library helps the student in grasping the basic concepts of computer vision. In the last four years we have conducted surveys to assess how much the students have improved their skills by using this library.

Graphical User Interface (GUI) for the representation of GM surfaces (using the NRTL model) and curves, including tie-lines and Hessian matrix

A single and very easy to use Graphical User Interface (GUI- MATLAB) based on the topological information contained in the Gibbs energy of mixing function has been developed as a friendly tool to check the coherence of NRTL parameters obtained in a correlation data procedure. Thus, the analysis of the GM/RT surface, the GM/RT for the binaries and the GM/RT in planes containing the tie lines should be necessary to validate the obtained parameters for the different models for correlating phase equlibrium data.

A Novel Prediction Method for Early Recognition of Global Human Behaviour in Image Sequences

Human behaviour recognition has been, and still remains, a challenging problem that involves different areas of computational intelligence. The automated understanding of people activities from video sequences is an open research topic in which the computer vision and pattern recognition areas have made big efforts. In this paper, the problem is studied from a prediction point of view. We propose a novel method able to early detect behaviour using a small portion of the input, in addition to the capabilities of it to predict behaviour from new inputs. Specifically, we propose a predictive method based on a simple representation of trajectories of a person in the scene which allows a high level understanding of the global human behaviour. The representation of the trajectory is used as a descriptor of the activity of the individual. The descriptors are used as a cue of a classification stage for pattern recognition purposes. Classifiers are trained using the trajectory representation of the complete sequence. However, partial sequences are processed to evaluate the early prediction capabilities having a specific observation time of the scene. The experiments have been carried out using the three different dataset of the CAVIAR database taken into account the behaviour of an individual. Additionally, different classic classifiers have been used for experimentation in order to evaluate the robustness of the proposal. Results confirm the high accuracy of the proposal on the early recognition of people behaviours.