bluuebunny/crossref_metadata_2025 huggingface dataset:
Approximating the distribution of population size in stochastic multiregional matrix models with fast migration
Authors: Alonso Juan Antonio , Sanz Luis
Published: November 2009

In this work we deal with a multiregional model in discrete time for an age-structured population which lives in an environment that changes randomly with time and is distributed in different spatial patches. In addition, and as is often the case in applications, we assume that migration is fast with respect to demography. Using approximate aggregation techniques we make use of the existence of different time scales in the model and reduce the dimension of the system obtaining a stochastic Leslie model in which the variables are the total population in each age class. Literature shows that, under reasonable conditions, the distribution of population size in matrix models with environmental stochasticity is asymptotically lognormal, and is characterized by two parameters, stochastic growth rate (s.g.r.) and scaled logarithmic variance (s.l.v.), that, in most practical cases, cannot be computed exactly. We show that the s.g.r. and the s.l.v. of the original multiregional model can be approximated by those corresponding to the reduced stochastic Leslie model, therefore simplifying its analysis. Moreover, we illustrate the usefulness of the reduction procedure by presenting some practical cases in which, although the explicit computation of the s.g.r. and the s.l.v. of the original multiregional model is not feasible, we can calculate its analogues for the reduced model.
A multiformalism and multiresolution modelling environment: application to the cardiovascular system and its regulation
Authors: Hernández Alfredo I. , Le Rolle Virginie , Defontaine Antoine , Carrault Guy
Published: November 2009

The role of modelling and simulation in the systemic analysis of living systems is now clearly established. Emerging disciplines, such as systems biology, and worldwide research actions, such as the Physiome Project or the Virtual Physiological Human, are based on an intensive use of modelling and simulation methodologies and tools. One of the key aspects in this context is to perform an efficient integration of various models representing different biological or physiological functions, at different resolutions, spanning through different scales. This paper presents a multiformalism modelling and simulation environment (M2SL) that has been conceived to ease model integration. A given model is represented as a set of coupled and atomic model components that may be based on different mathematical formalisms with heterogeneous structural and dynamical properties. A co-simulation approach is used to solve these hybrid systems. The pioneering model of the overall regulation of the cardiovascular system proposed by Guyton and co-workers in 1972 has been implemented under M2SL and a pulsatile ventricular model based on a time-varying elastance has been integrated in a multi-resolution approach. Simulations reproducing physiological conditions and using different coupling methods show the benefits of the proposed environment.
Micro-RNAs: viral genome and robustness of gene expression in the host
Authors: Demongeot Jacques , Drouet Emmanuel , Elena Adrien , Moreira Andrès , Rechoum Yassine , Sené Sylvain
Published: November 2009

For comparing RNA rings or hairpins with reference or random ring sequences, circular versions of distances and distributions like those of Hamming and Gumbel are needed. We define these circular versions and we apply these new tools to the comparison of RNA relics (such as micro-RNAs and tRNAs) with viral genomes that have coevolved with them. Then we show how robust are the regulation networks incorporating in their boundary micro-RNAs as sources or new feedback loops involving ubiquitous proteins like p53 (which is a micro-RNA transcription factor) or oligopeptides regulating protein translation. Eventually, we propose a new coevolution game between viral and host genomes.
Application of interval iterations to the entrainment problem in respiratory physiology
Authors: Demongeot Jacques , Waku Jules
Published: November 2009

We present here some theoretical and numerical results about interval iterations. We consider first an application of the interval iterations theory to the problem of entrainment in respiratory physiology for which the classical point iterations theory fails. Then, after a brief review of some of the main aspects of point iterations, we explain what is meant by the term ‘interval iterations’. It consists essentially in replacing in the point iterations the function to iterate by a set-valued map. We present both theoretical and numerical aspects of this new type of iterations and we observe the dynamical behaviours encountered, such as fixed intervals and interval limit cycles. The comparison between point and interval iterations is carried out with respect to a parameterε, which determines the thickness of a neighbourhood around the function to iterate. We will finally focus our attention on the Verhulst and Ricker functions largely used in population dynamics, which exhibit various asymptotic behaviours.
Place illusion and plausibility can lead to realistic behaviour in immersive virtual environments
Authors: Slater Mel
Published: November 2009

In this paper, I address the question as to why participants tend to respond realistically to situations and events portrayed within an immersive virtual reality system. The idea is put forward, based on the experience of a large number of experimental studies, that there are two orthogonal components that contribute to this realistic response. The first is ‘being there’, often called ‘presence’, the qualia of having a sensation of being in a real place. We call this place illusion (PI). Second, plausibility illusion (Psi) refers to the illusion that the scenario being depicted is actually occurring. In the case of both PI and Psi the participant knows for sure that they are not ‘there’ and that the events are not occurring. PI is constrained by the sensorimotor contingencies afforded by the virtual reality system. Psi is determined by the extent to which the system can produce events that directly relate to the participant, the overall credibility of the scenario being depicted in comparison with expectations. We argue that when both PI and Psi occur, participants will respond realistically to the virtual reality.
Perceiving emotion: towards a realistic understanding of the task
Authors: Cowie Roddy
Published: November 2009

A decade ago, perceiving emotion was generally equated with taking a sample (a still photograph or a few seconds of speech) that unquestionably signified an archetypal emotional state, and attaching the appropriate label. Computational research has shifted that paradigm in multiple ways. Concern with realism is key. Emotion generally colours ongoing action and interaction: describing that colouring is a different problem from categorizing brief episodes of relatively pure emotion. Multiple challenges flow from that. Describing emotional colouring is a challenge in itself. One approach is to use everyday categories describing states that are partly emotional and partly cognitive. Another approach is to use dimensions. Both approaches need ways to deal with gradual changes over time and mixed emotions. Attaching target descriptions to a sample poses problems of both procedure and validation. Cues are likely to be distributed both in time and across modalities, and key decisions may depend heavily on context. The usefulness of acted data is limited because it tends not to reproduce these features. By engaging with these challenging issues, research is not only achieving impressive results, but also offering a much deeper understanding of the problem.
Role of facial expressions in social interactions
Authors: Frith Chris
Published: November 2009

The expressions we see in the faces of others engage a number of different cognitive processes. Emotional expressions elicit rapid responses, which often imitate the emotion in the observed face. These effects can even occur for faces presented in such a way that the observer is not aware of them. We are also very good at explicitly recognizing and describing the emotion being expressed. A recent study, contrasting human and humanoid robot facial expressions, suggests that people can recognize the expressions made by the robot explicitly, but may not show the automatic, implicit response. The emotional expressions presented by faces are not simply reflexive, but also have a communicative component. For example, empathic expressions of pain are not simply a reflexive response to the sight of pain in another, since they are exaggerated when the empathizer knows he or she is being observed. It seems that we want people to know that we are empathic. Of especial importance among facial expressions are ostensive gestures such as the eyebrow flash, which indicate the intention to communicate. These gestures indicate, first, that the sender is to be trusted and, second, that any following signals are of importance to the receiver.
Future affective technology for autism and emotion communication
Authors: Picard Rosalind W.
Published: November 2009

People on the autism spectrum often experience states of emotional or cognitive overload that pose challenges to their interests in learning and communicating. Measurements taken from home and school environments show that extreme overload experienced internally, measured as autonomic nervous system (ANS) activation, may not be visible externally: a person can have a resting heart rate twice the level of non-autistic peers, while outwardly appearing calm and relaxed. The chasm between what is happening on the inside and what is seen on the outside, coupled with challenges in speaking and being pushed to perform, is a recipe for a meltdown that may seem to come ‘out of the blue’, but in fact may have been steadily building. Because ANS activation both influences and is influenced by efforts to process sensory information, interact socially, initiate motor activity, produce meaningful speech and more, deciphering the dynamics of ANS states is important for understanding and helping people on the autism spectrum. This paper highlights advances in technology that can comfortably sense and communicate ANS arousal in daily life, allowing new kinds of investigations to inform the science of autism while also providing personalized feedback to help individuals who participate in the research.
Promoting motivation with virtual agents and avatars: role of visual presence and appearance
Authors: Baylor Amy L.
Published: November 2009

Anthropomorphic virtual agents can serve as powerful technological mediators to impact motivational outcomes such as self-efficacy and attitude change. Such anthropomorphic agents can be designed as simulated
social models
in the Bandurian sense, providing social influence as virtual ‘role models’. Of particular value is the capacity for designing such agents as optimized social models for a target audience and context. Importantly, the
visual presence
and
appearance
of such agents can have a major impact on motivation and affect regardless of the underlying technical sophistication. Empirical results of different instantiations of agent presence and appearance are reviewed for both autonomous virtual agents and avatars that represent a user.
Effects of damping head movement and facial expression in dyadic conversation using real–time facial expression tracking and synthesized avatars
Authors: Boker Steven M. , Cohn Jeffrey F. , Theobald Barry-John , Matthews Iain , Brick Timothy R. , Spies Jeffrey R.
Published: November 2009

When people speak with one another, they tend to adapt their head movements and facial expressions in response to each others' head movements and facial expressions. We present an experiment in which confederates' head movements and facial expressions were motion tracked during videoconference conversations, an avatar face was reconstructed in real time, and naive participants spoke with the avatar face. No naive participant guessed that the computer generated face was not video. Confederates' facial expressions, vocal inflections and head movements were attenuated at 1 min intervals in a fully crossed experimental design. Attenuated head movements led to increased head nods and lateral head turns, and attenuated facial expressions led to increased head nodding in both naive participants and confederates. Together, these results are consistent with a hypothesis that the dynamics of head movements in dyadicconversation include a shared equilibrium. Although both conversational partners were blind to the manipulation, when apparent head movement of one conversant was attenuated, both partners responded by increasing the velocity of their head movements.
Designing for emotion (among other things)
Authors: Gaver William
Published: November 2009

Using computational approaches to emotion in design appears problematic for a range of technical, cultural and aesthetic reasons. After introducing some of the reasons as to why I am sceptical of such approaches, I describe a prototype we built that tried to address some of these problems, using sensor-based inferencing to comment upon domestic ‘well-being’ in ways that encouraged users to take authority over the emotional judgements offered by the system. Unfortunately, over two iterations we concluded that the prototype we built was a failure. I discuss the possible reasons for this and conclude that many of the problems we found are relevant more generally for designs based on computational approaches to emotion. As an alternative, I advocate a broader view of interaction design in which open-ended designs serve as resources for individual appropriation, and suggest that emotional experiences become one of several outcomes of engaging with them.
Modelling multimodal expression of emotion in a virtual agent
Authors: Pelachaud Catherine
Published: November 2009

Over the past few years we have been developing an expressive embodied conversational agent system. In particular, we have developed a model of multimodal behaviours that includes dynamism and complex facial expressions. The first feature refers to the qualitative execution of behaviours. Our model is based on perceptual studies and encompasses several parameters that modulate multimodal behaviours. The second feature, the model of complex expressions, follows a componential approach where a new expression is obtained by combining facial areas of other expressions. Lately we have been working on adding temporal dynamism to expressions. So far they have been designed statically, typically at their apex. Only full-blown expressions could be modelled. To overcome this limitation, we have defined a representation scheme that describes the temporal evolution of the expression of an emotion. It is no longer represented by a static definition but by a temporally ordered sequence of multimodal signals.
Why bodies? Twelve reasons for including bodily expressions in affective neuroscience
Authors: de Gelder Beatrice
Published: November 2009

Why bodies? It is rather puzzling that given the massive interest in affective neuroscience in the last decade, it still seems to make sense to raise the question ‘Why bodies’ and to try to provide an answer to it, as is the goal of this article. There are now hundreds of articles on human emotion perception ranging from behavioural studies to brain imaging experiments. These experimental studies complement decades of reports on affective disorders in neurological patients and clinical studies of psychiatric populations. The most cursory glance at the literature on emotion in humans, now referred to by the umbrella term of social and affective neuroscience, shows that over 95 per cent of them have used faces as stimuli. Of the remaining 5 per cent, a few have used scenes or auditory information including human voices, music or environmental sounds. But by far the smallest number has looked into whole-body expressions. As a rough estimate, a search on PubMed today, 1 May 2009, yields 3521 hits for emotion × faces, 1003 hits for emotion × music and 339 hits for emotion × bodies. When looking in more detail, the body × emotion category in fact yields a majority of papers on well-being, nursing, sexual violence or organ donation. But the number of cognitive and affective neuroscience studies of emotional body perception as of today is lower than 20.

Why then have whole bodies and bodily expressions not attracted the attention of researchers so far? The goal of this article is to contribute some elements for an answer to this question. I believe that there is something to learn from the historical neglect of bodies and bodily expressions. I will next address some historical misconceptions about whole-body perception, and in the process I intend not only to provide an impetus for this kind of work but also to contribute to a better understanding of the significance of the affective dimension of behaviour, mind and brain as seen from the vantage point of bodily communication. Subsequent sections discuss available evidence for the neurofunctional basis of facial and bodily expressions as well as neuropsychological and clinical studies of bodily expressions.
Can emotion recognition be taught to children with autism spectrum conditions?
Authors: Baron-Cohen Simon , Golan Ofer , Ashwin Emma
Published: November 2009

Children with autism spectrum conditions (ASC) have major difficulties in recognizing and responding to emotional and mental states in others' facial expressions. Such difficulties in empathy underlie their social-communication difficulties that form a core of the diagnosis. In this paper we ask whether aspects of empathy can be taught to young children with ASC. We review a study that evaluatedThe Transporters, an animated series designed to enhance emotion comprehension in children with ASC. Children with ASC (4–7 years old) watchedThe Transportersevery day for four weeks. Participants were tested before and after intervention on emotional vocabulary and emotion recognition at three levels of generalization. The intervention group improved significantly more than a clinical control group on all task levels, performing comparably to typical controls at time 2. The discussion centres on how vehicles as mechanical systems may be one key reason whyThe Transporterscaused the improved understanding and recognition of emotions in children with ASC. The implications for the design of autism-friendly interventions are also explored.
Computation of emotions in man and machines
Authors: Robinson Peter , el Kaliouby Rana
Published: November 2009

The importance of emotional expression as part of human communication has been understood since Aristotle, and the subject has been explored scientifically since Charles Darwin and others in the nineteenth century. Advances in computer technology now allow machines to recognize and express emotions, paving the way for improved human–computer and human–human communications.

Recent advances in psychology have greatly improved our understanding of the role of affect in communication, perception, decision-making, attention and memory. At the same time, advances in technology mean that it is becoming possible for machines to sense, analyse and express emotions. We can now consider how these advances relate to each other and how they can be brought together to influence future research in perception, attention, learning, memory, communication, decision-making and other applications.

The computation of emotions includes both recognition and synthesis, using channels such as facial expressions, non-verbal aspects of speech, posture, gestures, physiology, brain imaging and general behaviour. The combination of new results in psychology with new techniques of computation is leading to new technologies with applications in commerce, education, entertainment, security, therapy and everyday life. However, there are important issues of privacy and personal expression that must also be considered.
Affective loop experiences: designing for interactional embodiment
Authors: Höök Kristina
Published: November 2009

Involving our corporeal bodies in interaction can create strong affective experiences. Systems that both can be influenced by and influence users corporeally exhibit a use quality we name anaffective loopexperience. In an affective loop experience, (i) emotions are seen as processes, constructed in the interaction, starting from everyday bodily, cognitive or social experiences; (ii) the system responds in ways that pull the user into the interaction, touching upon end users' physical experiences; and (iii) throughout the interaction the user is an active, meaning-making individual choosing how to express themselves—the interpretation responsibility does not lie with the system. We have built several systems that attempt to create affective loop experiences with more or less successful results. For example,eMotolets users send text messages between mobile phones, but in addition to text, the messages also have colourful and animated shapes in the background chosen through emotion-gestures with a sensor-enabled stylus pen.Affective Diaryis a digital diary with which users can scribble their notes, but it also allows forbodily memorabiliato be recorded from body sensors mapping to users' movement and arousal and placed along a timeline. Users can see patterns in their bodily reactions and relate them to various events going on in their lives.

The experiences of building and deploying these systems gave us insights into design requirements for addressing affective loop experiences, such as how to design for turn-taking between user and system, how to create for ‘open’ surfaces in the design that can carry users' own meaning-making processes, how to combine modalities to create for a ‘unity’ of expression, and the importance of mirroring user experience in familiar ways that touch upon their everyday social and corporeal experiences.

But a more important lesson gained from deploying the systems is how emotion processes are co-constructed and experienced inseparable from all other aspects of everyday life. Emotion processes are part of our social ways of being in the world; they dye our dreams, hopes and bodily experiences of the world. If we aim to design for affective interaction experiences, we need to place them into this larger picture.
Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β
Authors: Sato Hanae , Maquat Lynne E.
Published: November 2009

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)–protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)–CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon–exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin β (IMPβ): Inhibiting the binding of IMPβ to the complex of CBC–IMPα at an mRNA cap using the IMPα IBB (IMPβ-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPβ and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured.
Endoreplication: polyploidy with purpose
Authors: Lee Hyun O. , Davidson Jean M. , Duronio Robert J.
Published: November 2009

A great many cell types are necessary for the myriad capabilities of complex, multicellular organisms. One interesting aspect of this diversity of cell type is that many cells in diploid organisms are polyploid. This is called endopolyploidy and arises from cell cycles that are often characterized as “variant,” but in fact are widespread throughout nature. Endopolyploidy is essential for normal development and physiology in many different organisms. Here we review how both plants and animals use variations of the cell cycle, termed collectively as endoreplication, resulting in polyploid cells that support specific aspects of development. In addition, we discuss briefly how endoreplication occurs in response to certain physiological stresses, and how it may contribute to the development of cancer. Finally, we describe the molecular mechanisms that support the onset and progression of endoreplication.
SetDB1 contributes to repression of genes encoding developmental regulators and maintenance of ES cell state
Authors: Bilodeau Steve , Kagey Michael H. , Frampton Garrett M. , Rahl Peter B. , Young Richard A.
Published: November 2009

Transcription factors that play key roles in regulating embryonic stem (ES) cell state have been identified, but the chromatin regulators that help maintain ES cells are less well understood. A high-throughput shRNA screen was used to identify novel chromatin regulators that influence ES cell state. Loss of histone H3 Lys 9 (H3K9) methyltransferases, particularly SetDB1, had the most profound effects on ES cells. Chromatin immunoprecipitation (ChIP) coupled with massively parallel DNA sequencing (ChIP-Seq) and functional analysis revealed that SetDB1 and histone H3K9-methylated nucleosomes occupy and repress genes encoding developmental regulators. These SetDB1-occupied genes are a subset of the “bivalent” genes, which contain nucleosomes with H3K4me3 (H3K4 trimethylation) and H3K27me3 modifications catalyzed by Trithorax and Polycomb group proteins, respectively. These genes are subjected to repression by both Polycomb group proteins and SetDB1, and loss of either regulator can destabilize ES cell state.
Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity
Authors: Lee Hojoon X. , Mendes Fabio A. , Plouhinec Jean-Louis , De Robertis Edward M.
Published: November 2009

In Xenopus embryos, a dorsal–ventral patterning gradient is generated by diffusing Chordin/bone morphogenetic protein (BMP) complexes cleaved by BMP1/Tolloid metalloproteinases in the ventral side. We developed a new BMP1/Tolloid assay using a fluorogenic Chordin peptide substrate and identified an unexpected negative feedback loop for BMP4, in which BMP4 inhibits Tolloid enzyme activity noncompetitively. BMP4 binds directly to the CUB (Complement 1r/s, Uegf [a sea urchin embryonic protein] and BMP1) domains of BMP1 and Drosophila Tolloid with high affinity. Binding to CUB domains inhibits BMP4 signaling. These findings provide a molecular explanation for a long-standing genetical puzzle in which antimorphic Drosophila tolloid mutant alleles displayed anti-BMP effects. The extensive Drosophila genetics available supports the relevance of the interaction described here at endogenous physiological levels. Many extracellular proteins contain CUB domains; the binding of CUB domains to BMP4 suggests a possible general function in binding transforming growth factor-β (TGF-β) superfamily members. Mathematical modeling indicates that feedback inhibition by BMP ligands acts on the ventral side, while on the dorsal side the main regulator of BMP1/Tolloid enzymatic activity is the binding to its substrate, Chordin.
A kiss of death—proteasome-mediated membrane fusion and programmed cell death in plant defense against bacterial infection: Figure 1.
Authors: Pajerowska-Mukhtar Karolina , Dong Xinnian
Published: November 2009

Eukaryotes have evolved various means for controlled and organized cellular destruction, known as programmed cell death (PCD). In plants, PCD is a crucial regulatory mechanism in multiple physiological processes, including terminal differentiation, senescence, and disease resistance. In this issue of Genes & Development, Hatsugai and colleagues (pp. 2496–2506) demonstrate a novel plant defense strategy to trigger bacteria-induced PCD, involving proteasome-dependent tonoplast and plasma membrane fusion followed by discharge of vacuolar antimicrobial and death-inducing contents into the apoplast.
Subtraction by addition: domesticated transposases in programmed DNA elimination: Figure 1.
Authors: Motl Jason A. , Chalker Douglas L.
Published: November 2009

The ciliate Paramecium tetraurelia must eliminate ∼60,000 short sequences from its genome to generate uninterrupted coding sequences in its somatic macronucleus. In this issue of Genes & Development, Baudry and colleagues (pp. 2478–2483) identify the protein that excises these noncoding sequences: a domesticated piggyBac transposase that has been adapted to remove what are likely the remnants of transposon insertions. This new study reveals how addition of a transposase to small RNA-directed silencing machinery can guide major genome reorganization.
PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements in the ciliate Paramecium tetraurelia
Authors: Baudry Céline , Malinsky Sophie , Restituito Matthieu , Kapusta Aurélie , Rosa Sarah , Meyer Eric , Bétermier Mireille
Published: November 2009

Programmed genome rearrangements drive functional gene assembly in ciliates during the development of the somatic macronucleus. The elimination of germline sequences is directed by noncoding RNAs and is initiated by DNA double-strand breaks, but the enzymes responsible for DNA cleavage have not been identified. We show here that PiggyMac (Pgm), a domesticated piggyBac transposase, is required for these rearrangements in Paramecium tetraurelia. A GFP-Pgm fusion localizes in developing macronuclei, where rearrangements take place, and RNAi-mediated silencing of PGM abolishes DNA cleavage. This is the first in vivo evidence suggesting an essential endonucleolytic function of a domesticated piggyBac transposase.
The Hawaiian Bobtail Squid (Euprymna scolopes): A Model to Study the Molecular Basis of Eukaryote-Prokaryote Mutualism and the Development and Evolution of Morphological Novelties in Cephalopods
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert
Published: November 2009

INTRODUCTION

The Hawaiian bobtail squid, Euprymna scolopes, is a cephalopod whose small size, short lifespan, rapid growth, and year-round availability make it suitable as a model organism. E. scolopes is studied in three principal contexts: (1) as a model of cephalopod development; (2) as a model of animal-bacterial symbioses; and (3) as a system for studying adaptations of tissues that interact with light. E. scolopes embryos can be obtained continually and can be reared in the laboratory over an entire generation. The embryos and protective chorions are optically clear, facilitating in situ developmental observations, and can be manipulated experimentally. Many molecular protocols have been developed for studying E. scolopes development. This species is best known, however, for its symbiosis with the luminous marine bacterium Vibrio fischeri and has been used to study determinants of symbiont specificity, the influence of symbiosis on development of the squid light organ, and the mechanisms by which a stable association is achieved. Both partners can be grown independently under laboratory conditions, a feature that offers the unusual opportunity to manipulate the symbiosis experimentally. Molecular and genetic tools have been developed for V. fischeri, and a large expressed sequence tag (EST) database is available for the host symbiotic tissues. Additionally, comparisons between light organ form and function to those of the eye can be made. Both types of tissue interact with light, but have divergent embryonic development. As such, they offer an opportunity to study the molecular basis for the evolution of morphological novelties.
True Yams(Dioscorea):A Biological and Evolutionary Link between Eudicots and Grasses
Authors: Mignouna Hodeba D. , Abang Mathew M. , Asiedu Robert , Geeta R.
Published: November 2009

INTRODUCTION

Dioscorea(true yams) is a large genus that contains species important as food (with edible tubers) or as sources of bioactive substances used in a range of applications.Dioscoreais a major staple food in many parts of the world, especially in West Africa and the Pacific islands, and serves as a famine food in many regions. It is a critically important but neglected crop, which is likely to increase in importance as climate change leads to necessary changes in global food systems. It is a herbaceous, climbing, tropical monocot that looks rather like a dicot, and is part of a lineage that is relatively closely related to the phylogenetically derived group containing the grasses. Therefore, it represents an important biological link between the eudicots and grasses--groups that contain all the model flowering plant species--and has the potential to fill gaps in our knowledge of plant biology and evolution. Yams also offer us the possibility to gain new insights into processes such as tuberization and sex determination, which cannot be studied in current model organisms. This combination of rising importance due to its socioeconomic significance and interesting biology and evolutionary position justify its potential as a model organism. This potential remains to be harnessed, and much of the current work on yam is directed toward its role as a food crop. This aspect will remain important, but its potential for answering questions of basic biological interest will be a major motivation for researchers interested in this organism.
The Embryonic Development of the Hawaiian Bobtail Squid (Euprymna scolopes)
Authors: Lee Patricia N. , Callaerts Patrick , de Couet H. Gert
Published: November 2009

INTRODUCTION

A staging series based on easily distinguishable morphological features is a basic and necessary tool for developmental studies. It provides a consistent reference for comparisons between independent studies, negates the need to know when fertilization occurred, allows correlation of the phase of development with the time of development (to facilitate collection of embryos at specific stages), and allows comparisons between species. Given the growing interest in Hawaiian bobtail squid (Euprymna scolopes) as a contemporary cephalopod developmental system, this article provides a detailed survey of E. scolopes embryogenesis from cleavage through hatching under controlled environmental conditions, including detailed descriptions of externally visible morphological features that are easily distinguished in either live or freshly fixed embryos under a dissecting microscope. Photomicrographs are also provided to aid in the accurate and rapid staging of E. scolopes embryos.
Labeling Pinocytotic Vesicles and Cytoplasm with Fluorescent Dextrans or Ficolls for Imaging
Authors: Chazotte Brad
Published: November 2009

INTRODUCTION

The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, in cell biology, the study of organization and structure/function relationships is of great importance. Dextran, a high-molecular-weight poly-D-glucose, and ficoll, a synthetic polymer of epichlorhydrin and sucrose, are electroneutral, hydrophilic polysaccharides whose size can be varied. This variability in size coupled with their membrane impermeability can make them useful for studying fluid-phase pinocytosis, the size of membrane pores (e.g., nuclear membrane pores), or the environmental conditions and size of a cell compartment. This article presents a method for labeling live or fixed cells with substituted forms of dextran or ficoll attached to a fluorescent conjugate. In the event that the desired probe is not available commercially, this article also provides a method for covalently attaching a fluorophore to substituted polysaccharide.
Phenotypic Analysis of Arabidopsis Mutants: Root Elongation under Salt/Hormone-Induced Stress
Authors: Lee Byeong-ha , Zhu Jian-Kang
Published: November 2009

INTRODUCTION

Abiotic stress, such as high salt or low temperature, adversely affects plant growth and development. Salt stress inhibits seed germination, retards plant growth, and accelerates senescence. Freezing or drought stress can cause cell damage and plant death. The following parameters can be used to evaluate plant tolerance to salt, drought, or freezing stress: root elongation (described here), fresh weight gain, seed germination, electrolyte leakage, or water loss measurement. Several stress mutants have been characterized using these tests, including hos1 and hos2, which show higher expression of some stress-regulated genes when exposed to low-temperature stress; hos5, which shows higher expression of some stress-regulated genes under abscisic acid (ABA) and salt treatments; sfr mutants, which are deficient in freezing tolerance; and eskimo1, which is constitutively freezing-tolerant. To determine whether a mutant shows altered response to osmotic stress or to specific ions, various concentrations of salts can be used. Mannitol can also be used to impose osmotic stress, and ABA can be used to impose hormone stress. Among the salts used in this protocol, Li+ is considered a toxic analog of Na+, whereas Cs+ is a toxic cation related to K+. The levels of stress suggested in this protocol may need to be adjusted, depending on the ecotype and growth conditions used.
Preparation of Soluble GST Fusion Proteins
Authors: Rebay Ilaria , Fehon Richard G.
Published: November 2009

INTRODUCTION

A wide variety of plasmid vectors are commercially available for the production of fusion proteins in bacterial cells. Most are also designed to incorporate a tag that allows affinity purification of the expressed fusion protein from bacterial cell extracts. The most commonly used are vectors that incorporate a portion of the glutathione-S-transferase (GST) enzyme that is able to bind to immobilized glutathione and vectors that use a polyhistidine tag which binds immobilized nickel ions with high affinity. This protocol describes preparation of a soluble GST fusion protein, isolated using glutathione agarose beads.
Imaging Synaptogenesis by Measuring Accumulation of Synaptic Proteins: Figure 1.
Authors: Dean Camin , Scheiffele Peter
Published: November 2009

INTRODUCTION

Synapse formation and modification involve the successive recruitment of pre- and post-synaptic signaling molecules. The procedure presented here investigates the function of specific synaptic factors in gain- and loss-­of-function experiments by measuring protein accumulation at synapses using quantitative immunohistochemistry, confocal microscopy, and image analysis. Although the procedure is described for dissociated cultures of hippocampal neurons, it can be extended to various neuronal cell types in culture.
Imaging Action Potentials with Calcium Indicators: Figure 1.
Authors: MacLean Jason N. , Yuste Rafael
Published: November 2009

INTRODUCTION

The understanding of neuronal circuits has been, and will continue to be, greatly advanced by the simultaneous imaging of action potentials in neuronal ensembles. This protocol describes “bulk” loading of brain slices with acetoxymethyl (AM) ester calcium indicators in order to monitor action potential activity in large populations of neurons simultaneously. The imaging of calcium influx into neurons provides an indirect, but accurate, measure of action potential generation in individual neurons. Single-cell resolution, and thus the easy identification of every active cell, is the key advantage of the technique.
Indirect Immunofluorescence Labeling in the Yeast Saccharomyces cerevisiae
Authors: Silver Pamela
Published: November 2009

INTRODUCTION

The budding yeast Saccharomyces cerevisiae is an ideal model system for the initial characterization of novel genes and their corresponding gene products. Genetic analysis is straightforward, and sophisticated cell biological methods are available. Furthermore, only in a genetically tractable organism is it possible to use epitope-tagged proteins to the best advantage. In S. cerevisiae, it is a trivial matter to determine whether a modified version of a protein is functional. This is accomplished by testing whether the tagged protein can complement a null phenotype or rescue a conditional phenotype. Methods such as indirect immunofluorescence and the use of green fluorescent protein (GFP) allow the investigator to correlate the intracellular localization of the protein with its function in vivo. This article includes a detailed protocol for performing indirect immunofluorescence with S. cerevisiae. Cells are grown exponentially and then fixed. After fixation, an enzyme is used to remove the cell wall, and the cells are adhered to slides. Subsequent steps involve the application of primary and secondary antibodies and the final processing of the slide.
Bimolecular Affinity Purification (BAP): Tandem Affinity Purification Using Two Protein Baits: Figure 1.
Authors: Maine Gabriel N. , Gluck Nathan , Zaidi Iram W. , Burstein Ezra
Published: November 2009

INTRODUCTION

The tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes. While affinity purification is relatively easy to perform, nonspecific protein interactions can plague the identification of true interacting partners of the given bait utilized in the purification. To alleviate this problem, two sequential affinity purification steps are employed in the TAP procedure. Since its inception in yeast, TAP has gone through many adaptations and has been employed multiple times in diverse organisms, including mammalian systems. In all these approaches, two out of many possible affinity moieties are employed and are usually expressed as a fusion polypeptide in the amino or carboxyl-terminal region of the protein bait. In this protocol, we describe a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, which we refer to as bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components.
Preparation of Genomic DNA from Hawaiian Bobtail Squid (Euprymna scolopes) Tissue by Cesium Chloride Gradient Centrifugation
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert
Published: November 2009

INTRODUCTION

This procedure describes the extraction of genomic DNA from adult bobtail squid (Euprymna scolopes) tissues by cesium chloride (CsCl) gradient centrifugation. There are numerous generic methods and commercial kits for the preparation of genomic DNA based on proteolytic digestion of chromatin components, followed by selective binding of nucleic acids to ion-exchange affinity media, but many of these do not yield DNA that can be readily restricted. Also, molluscan tissues contain mucopolysaccharides, which tend to copurify with DNA under certain conditions. Although nucleic acids prepared this way can serve as a template for polymerase chain reaction (PCR), other enzymatic modifications of nucleic acids are inhibited by these contaminants. The method described here yields high-molecular-weight DNA that can be readily restricted for Southern hybridization. The procedure uses brain tissue under the assumption that its genome is unlikely to be rearranged in any way, has a high nucleic acid:protein ratio, and avoids potential sources of enzymatic contaminants and parasites from the intestinal sac. However, the method can be applied to other tissue sources and works well with other species. The purification of DNA by gradient centrifugation is an established method based on the specific buoyant density of double-stranded nucleic acids and the ability of CsCl solutions to form a salt gradient in a centrifugal field. It can also be adapted to the purification of RNA, which has a higher buoyant density than DNA. Unfortunately, this method is somewhat involved and expensive and produces large amounts of ethidium bromide waste.
Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled Riboprobes: I. DNA Template Preparation and In Vitro Transcription of Riboprobes
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert
Published: November 2009

INTRODUCTION

Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of a DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess, unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the techniques for preparing RNA probes for whole-mount in situ hybridization in Hawaiian bobtail squid (Euprymna scolopes) embryos from linearized plasmid DNA or polymerase chain reaction (PCR) products.
Whole-Mount In Situ Hybridization of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos with DIG-Labeled Riboprobes: II. Embryo Preparation, Hybridization, Washes, and Immunohistochemistry
Authors: Lee Patricia N. , McFall-Ngai Margaret J. , Callaerts Patrick , de Couet H. Gert
Published: November 2009

INTRODUCTION

Whole-mount in situ hybridization is a technique used to localize and visualize specific gene transcripts in whole embryos by hybridizing labeled RNA probes complementary to the sequence of interest. A digoxigenin (DIG)-labeled riboprobe synthesized during in vitro transcription through the incorporation of DIG-labeled UTP is hybridized to the target sequence under stringent conditions, and excess unhybridized probe is removed during a series of washes. The location of the labeled riboprobe, and thus the mRNA sequence of interest, is then visualized by immunohistochemistry. This protocol outlines the steps involved in preparing Hawaiian bobtail squid (Euprymna scolopes) embryos, hybridizing a DIG-labeled riboprobe in whole-mount embryos, and visualizing the labeled RNA colorimetrically using an alkaline-phosphatase-conjugated anti-DIG antibody.
Culture of Hawaiian Bobtail Squid (Euprymna scolopes) Embryos and Observation of Normal Development
Authors: Lee Patricia N. , Callaerts Patrick , de Couet H. Gert
Published: November 2009

INTRODUCTION

The ability to rear Hawaiian bobtail squid (Euprymna scolopes) embryos under controlled environmental conditions is a basic and necessary tool for developmental studies. It negates the need to know when fertilization occurred, allows correlation of the phase of development with the time of development (thereby facilitating collection of embryos at specific stages), and allows comparisons between cephalopod species. Embryonic development in E. scolopes is robust over a range of temperatures, is relatively rapid (~21 d), and proceeds normally under laboratory conditions at ambient temperature (27°C-29°C). Here we present methods for maintaining E. scolopes embryos in culture from cleavage through hatching, as well as observing and recording live or freshly fixed embryos under a dissecting microscope.