bluuebunny/crossref_metadata_2025 huggingface dataset:
Whole cell mechanics of contractile fibroblasts: relations between effective cellular and extracellular matrix moduli
Authors: Marquez J. Pablo , Elson Elliot L. , Genin Guy M.
Published: January 2010

While much is known about the subcellular structures responsible for the mechanical functioning of a contractile fibroblast, debate exists about how these components combine to endow a cell with its form and mechanical function. We present an analysis of mechanical characterization experiments performed on bio-artificial tissue constructs, which we believe serve as a more realistic testing environment than two-dimensional cell culture. These model tissues capture many features of real tissues with the advantage that they can be engineered to model different physiological and pathological characteristics. We study here a model tissue consisting of reconstituted type I collagen and varying concentrations of activated contractile fibroblasts that is relevant to modelling different stages of wound healing. We applied this system to assess how cell and extracellular matrix (ECM) mechanics vary with cell concentration. Short-term and long-term moduli of the ECM were estimated through analytical and numerical analysis of two-phase elastic solids containing cell-shaped voids. The relative properties of cells were then deduced from the results of numerical analyses of two-phase elastic solids containing mechanically isotropic cells of varying modulus. With increasing cell concentration, the short-term and long-term tangent moduli of the reconstituted collagen ECM increased sharply from a baseline value, while those of the cells decreased monotonically.
Floral adaptation and diversification under pollen limitation
Authors: Harder Lawrence D. , Aizen Marcelo A.
Published: January 2010

Pollen limitation (PL) of seed production creates unique conditions for reproductive adaptation by angiosperms, in part because, unlike under ovule or resource limitation, floral interactions with pollen vectors can contribute to variation in female success. Although the ecological and conservation consequences of PL have received considerable attention in recent times, its evolutionary implications are poorly appreciated. To identify general influences of PL on reproductive adaptation compared with those under other seed-production limits and their implications for evolution in altered environments, we derive a model that incorporates pollination and post-pollination aspects of PL. Because PL always favours increased ovule fertilization, even when population dynamics are not seed limited, it should pervasively influence selection on reproductive traits. Significantly, under PL the intensity of inbreeding does not determine whether outcrossing or autonomous selfing can evolve, although it can affect which response is most likely. Because the causes of PL are multifaceted in both natural and anthropogenically altered environments, the possible outcrossing solutions are diverse and context dependent, which may contribute to the extensive variety of angiosperm reproductive characteristics. Finally, the increased adaptive options available under PL may be responsible for positive global associations between it and angiosperm diversity.
Diversity in obscurity: fossil flowers and the early history of angiosperms
Authors: Friis Else Marie , Pedersen Kaj Raunsgaard , Crane Peter R.
Published: January 2010

In the second half of the nineteenth century, pioneering discoveries of rich assemblages of fossil plants from the Cretaceous resulted in considerable interest in the first appearance of angiosperms in the geological record. Darwin's famous comment, which labelled the ‘rapid development’ of angiosperms an ‘abominable mystery’, dates from this time. Darwin and his contemporaries were puzzled by the relatively late, seemingly sudden and geographically widespread appearance of modern-looking angiosperms in Late Cretaceous floras. Today, the early diversification of angiosperms seems much less ‘rapid’. Angiosperms were clearly present in the Early Cretaceous, 20–30 Myr before they attained the level of ecological dominance reflected in some mid-Cretaceous floras, and angiosperm leaves and pollen show a distinct pattern of steadily increasing diversity and complexity through this interval. Early angiosperm fossil flowers show a similar orderly diversification and also provide detailed insights into the changing reproductive biology and phylogenetic diversity of angiosperms from the Early Cretaceous. In addition, newly discovered fossil flowers indicate considerable, previously unrecognized, cryptic diversity among the earliest angiosperms known from the fossil record. Lineages that today have an herbaceous or shrubby habit were well represented. Monocotyledons, which have previously been difficult to recognize among assemblages of early fossil angiosperms, were also diverse and prominent in many Early Cretaceous ecosystems.
The evolution of floral biology in basal angiosperms
Authors: Endress Peter K.
Published: January 2010

In basal angiosperms (including ANITA grade, magnoliids, Choranthaceae, Ceratophyllaceae) almost all bisexual flowers are dichogamous (with male and female functions more or less separated in time), and nearly 100 per cent of those are protogynous (with female function before male function). Movements of floral parts and differential early abscission of stamens in the male phase are variously associated with protogyny. Evolution of synchronous dichogamy based on the day/night rhythm and anthesis lasting 2 days is common. In a few clades in Magnoliales and Laurales heterodichogamy has also evolved. Beetles, flies and thrips are the major pollinators, with various degrees of specialization up to large beetles and special flies in some large-flowered Nymphaeaceae, Magnoliaceae, Annonaceae and Aristolochiaceae. Unusual structural specializations are involved in floral biological adaptations (calyptras, inner staminodes, synandria and food bodies, and secretory structures on tepals, stamens and staminodes). Numerous specializations that are common in monocots and eudicots are absent in basal angiosperms. Several families are poorly known in their floral biology.
The evolution and loss of oil-offering flowers: new insights from dated phylogenies for angiosperms and bees
Authors: Renner S. S. , Schaefer H.
Published: January 2010

The interactions between bees that depend on floral oil for their larvae and flowers that offer oil involve an intricate mix of obligate and facultative mutualisms. Using recent phylogenies, new data on oil-offering Cucurbitaceae, and molecular-dating, we ask when and how often oil-offering flowers and oil-foraging bees evolved, and how frequently these traits were lost in the cause of evolution. Local phylogenies and an angiosperm-wide tree show that oil flowers evolved at least 28 times and that floral oil was lost at least 36–40 times. The oldest oil flower systems evolved shortly after the K/T boundary independently in American Malpighiaceae, tropical African Cucurbitaceae and LaurasianLysimachia(Myrsinaceae); the ages of the South African oil flower/oil bee systems are less clear. Youngest oil flower clades includeCalceolaria(Calceolariaceae), Iridaceae,Krameria(Krameriaceae) and numerous Orchidaceae, many just a few million years old. In bees, oil foraging evolved minimally seven times and dates back to at least 56 Ma (Ctenoplectra) and 53 Ma (Macropis). The co-occurrence of older and younger oil-offering clades in three of the four geographical regions (but not the Holarctic) implies that oil-foraging bees acquired additional oil hosts over evolutionary time. Such niche-broadening probably started with exploratory visits to flowers resembling oil hosts in scent or colour, as suggested by several cases of Muellerian or Batesian mimicry involving oil flowers.
Aquilegiaas a model system for the evolution and ecology of petals
Authors: Kramer Elena M. , Hodges Scott A.
Published: January 2010

The ranunculid genusAquilegiaholds extraordinary promise as a model system for investigating a wide range of questions relating to the evolution and ecology of petals. New genetic and genomic resources, including an extensive EST database, BAC libraries and physical maps, as well as virus-induced gene silencing are facilitating this research on multiple fronts. At the developmental genetic level,Aquilegiahas been important for elucidating the developmental programme for specifying petals and petaloid characteristics. Data suggest that duplication events among the petal and stamen identity genes have resulted in sub- and neofunctionalization. This expansion of gene function does not include the petaloidy ofAquilegiasepals, however, which does not depend on the same loci that control identity of the second whorl petals. Of special interest is the elaboration of the petal into a nectar spur, a major innovation for the genus. Intra- and interspecific variation in the shape and colour of petals, especially the spurs, has been shown to be adaptative for different pollinators. Thus, understanding the genetic basis of these traits will help us connect the ecological interactions driving speciation with the genetic changes responsible for remodelling morphology. Progress in this area has focused on the multiple, parallel transitions in flower colour and nectar spur length across the genus. For flower colour, upstream transcription factors appear to be primarily targets of natural selection. Thus research inAquilegiaspans the initial evolution of petals and petaloidy to the diversification of petal morphology to the ecological basis of petal form, thereby providing a comprehensive picture of the evolutionary biology of this critical angiosperm feature.
Riding across the selection landscape: fitness consequences of annual variation in reproductive characteristics
Authors: Tremblay Raymond L. , Ackerman James D. , Pérez Maria-Eglée
Published: January 2010

Evolutionary models estimating phenotypic selection in character size usually assume that the character is invariant across reproductive bouts. We show that variation in the size of reproductive traits may be large over multiple events and can influence fitness in organisms where these traits are produced anew each season. With data from populations of two orchid species,Caladenia validaandTolumnia variegata, we used Bayesian statistics to investigate the effect on the distribution in fitness of individuals when the fitness landscape is not flat and when characters vary across reproductive bouts. Inconsistency in character size across reproductive periods within an individual increases the uncertainty of mean fitness and, consequently, the uncertainty in individual fitness. The trajectory of selection is likely to be muddled as a consequence of variation in morphology of individuals across reproductive bouts. The frequency and amplitude of such changes will certainly affect the dynamics between selection and genetic drift.
The evolutionary-developmental analysis of plant microRNAs
Authors: Jasinski Sophie , Vialette-Guiraud Aurélie C. M. , Scutt Charles P.
Published: January 2010

MicroRNAs (miRNAs) control many important aspects of plant development, suggesting these molecules may also have played key roles in the evolution of developmental processes in plants. However, evolutionary-developmental (evo-devo) studies of miRNAs have been held back by technical difficulties in gene identification. To help solve this problem, we have developed a two-step procedure for the efficient identification of miRNA genes in any plant species. As a test case, we have studied the evolution of the
MIR164
family in the angiosperms. We have identified novel
MIR164
genes in three species occupying key phylogenetic positions and used these, together with published sequence data, to partially reconstruct the evolution of the
MIR164
family since the last common ancestor of the extant flowering plants. We use our evolutionary reconstruction to discuss potential roles for
MIR164
genes in the evolution of leaf shape and carpel closure in the angiosperms. The techniques we describe may be applied to any miRNA family and should thus enable plant evo-devo to begin to investigate the contributions miRNAs have made to the evolution of plant development.
Spred1, a negative regulator of Ras–MAPK–ERK, is enriched in CNS germinal zones, dampens NSC proliferation, and maintains ventricular zone structure
Authors: Phoenix Timothy N. , Temple Sally
Published: January 2010

Neural stem cells (NSCs) have great potential for self-renewal, which must be tightly regulated to generate appropriate cell numbers during development and to prevent tumor formation. The Ras–MAPK–ERK pathway affects mitogen-stimulated proliferation, and negative regulators are likely to be important for keeping self-renewal in check. Sprouty-related protein with an EVH1 domain (Spred1) is a recently discovered negative Ras–MAPK–ERK regulator linked to a neurofibromatosis 1 (NF-1)-like human syndrome; however, its role in CNS development has not been explored. We show that Spred1 is highly enriched in CNS germinal zones during neurogenesis. Spred1 knockdown increases NSC self-renewal and progenitor proliferation cell-autonomously, and overexpression causes premature differentiation. Surprisingly, Spred1 knockdown in vivo in the embryonic mouse forebrain frequently resulted in periventricular heterotopia, developmental abnormalities often associated with mutations in genes in the vesicular trafficking pathway that cause disruption of germinal zones and impair cell migration. In cortical progenitor cells, Spred1 localizes within distinct vesicles, indicating a potential role in transport. Spred1 knockdown gradually leads to disruption of the apical ventricular zone and loss of radial glia alignment. This impairs late neuronal migration, resulting in the formation of periventricular masses. Thus, Spred1 is critical for normal cortical development, as it modulates progenitor self-renewal/proliferation and helps maintain the integrity and organization of germinal zones.
A coordinated phosphorylation by Lats and CK1 regulates YAP stability through SCFβ-TRCP
Authors: Zhao Bin , Li Li , Tumaneng Karen , Wang Cun-Yu , Guan Kun-Liang
Published: January 2010

The Yes-associated protein (YAP) transcription coactivator is a key regulator of organ size and a candidate human oncogene. YAP is inhibited by the Hippo pathway kinase cascade, at least in part via phosphorylation of Ser 127, which results in YAP 14–3–3 binding and cytoplasmic retention. Here we report that YAP is phosphorylated by Lats on all of the five consensus HXRXXS motifs. Phosphorylation of Ser 381 in one of them primes YAP for subsequent phosphorylation by CK1δ/ɛ in a phosphodegron. The phosphorylated phosphodegron then recruits the SCFβ-TRCP E3 ubiquitin ligase, which catalyzes YAP ubiquitination, ultimately leading to YAP degradation. The phosphodegron-mediated degradation and the Ser 127 phosphorylation-dependent translocation coordinately suppress YAP oncogenic activity. Our study identified CK1δ/ɛ as new regulators of YAP and uncovered an intricate mechanism of YAP regulation by the Hippo pathway via both S127 phosphorylation-mediated spatial regulation (nuclear–cytoplasmic shuttling) and the phosphodegron-mediated temporal regulation (degradation).
Genetic and biochemical definition of the Hedgehog receptor
Authors: Zheng Xiaoyan , Mann Randall K. , Sever Navdar , Beachy Philip A.
Published: January 2010

Although the transporter-like protein Patched (Ptc) is genetically implicated in reception of the extracellular Hedgehog (Hh) protein signal, a clear definition of the Hh receptor is complicated by the existence of additional Hh-binding proteins and, in Drosophila, by the lack of physical evidence for direct binding of Hh to Ptc. Here we show that activity of Ihog (Interference hedgehog), or of its close relative Boi (Brother of Ihog), is absolutely required for Hh biological response and for sequestration of the Hh protein to limit long-range signaling. We demonstrate that Ihog interacts directly with Ptc, is required for presentation of Ptc on the cell surface, and that Ihog and Ptc are both required for high-affinity Hh binding. On the basis of their joint roles in ligand binding, signal transduction, and receptor trafficking, we conclude that Ihog and Ptc together constitute the Drosophila Hh receptor.
Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNAiMet binding to the ribosome
Authors: Saini Adesh K. , Nanda Jagpreet S. , Lorsch Jon R. , Hinnebusch Alan G.
Published: January 2010

eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNAiMet) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE1) and SE2, was found to stimulate recruitment of Met-tRNAiMet in the ternary complex (TC) with eIF2·GTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI1 and SI2, reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNAiMet in the P site for AUG selection.
Leaping forks at inverted repeats: Figure 1.
Authors: Branzei Dana , Foiani Marco
Published: January 2010

Genome rearrangements are often associated with genome instability observed in cancer and other pathological disorders. Different types of repeat elements are common in genomes and are prone to instability. S-phase checkpoints, recombination, and telomere maintenance pathways have been implicated in suppressing chromosome rearrangements, but little is known about the molecular mechanisms and the chromosome intermediates generating such genome-wide instability. In the December 15, 2009, issue of Genes & Development, two studies by Paek and colleagues (2861–2875) and Mizuno and colleagues (pp. 2876–2886), demonstrate that nearby inverted repeats in budding and fission yeasts recombine spontaneously and frequently to form dicentric and acentric chromosomes. The recombination mechanism underlying this phenomenon does not appear to require double-strand break formation, and is likely caused by a replication mechanism involving template switching.
Switching the mechanism of mating type switching: a domesticated transposase supplants a domesticated homing endonuclease
Authors: Rusche Laura N. , Rine Jasper
Published: January 2010

Programmed DNA rearrangements are critical for the development of many organisms and, intriguingly, can be catalyzed by domesticated mobile genetic elements. In this issue of Genes & Development, Barsoum and colleagues (pp. 33–44) demonstrate that, in the budding yeast Kluyveromyces lactis, a DNA rearrangement associated with mating type switching requires a domesticated transposase and occurs through a mechanism distinct from that in the related yeast, Saccharomyces cerevisiae. Thus, mechanisms for mating type switching have evolved multiple times, indicating the relative ease with which mobile genetic elements can be captured.
Paramecium tetraurelia: The Renaissance of an Early Unicellular Model
Authors: Beisson Janine , Bétermier Mireille , Bré Marie-Hélène , Cohen Jean , Duharcourt Sandra , Duret Laurent , Kung Ching , Malinsky Sophie , Meyer Eric , Preer John R. , Sperling Linda
Published: January 2010

INTRODUCTION

Paramecium tetraurelia is a widely distributed, free-living unicellular organism that feeds on bacteria and can easily be cultured in the laboratory. Its position within the phylum Ciliophora, remote from the most commonly used models, offers an interesting perspective on the basic cellular and molecular processes of eukaryotic life. Its large size and complex cellular organization facilitate morphogenetic studies of conserved structures, such as cilia and basal bodies, as well as electrophysiological studies of swimming behavior. Like all ciliates, P. tetraurelia contains two distinct types of nuclei, the germline micronucleus (MIC) and the somatic macronucleus (MAC), which differentiate from copies of the zygotic nucleus after fertilization. The sexual cycle can be managed by controlling food uptake, allowing the study of a developmentally regulated differentiation program in synchronous cultures. Spectacular genome rearrangements occur during the development of the somatic macronucleus. Their epigenetic control by RNA-mediated homology-dependent mechanisms, which might underlie long-known cases of non-Mendelian inheritance, provides evolutionary insight into the diversity of small RNA pathways involved in genome regulation. Being endowed with two alternative modes of sexual reproduction (conjugation and autogamy), P. tetraurelia is ideally suited for genetic analyses, and the recent sequencing of its macronuclear genome revealed one of the largest numbers of genes in any eukaryote. Together with the development of new molecular techniques, including complementation cloning and an easily implemented technique for reverse genetics based on RNA interference (RNAi), these features make P. tetraurelia a very attractive unicellular model.
Measurement of Free Ca2+ Concentration in the Lumen of Neuronal Endoplasmic Reticulum
Authors: Solovyova Natasha , Verkhratsky Alexei
Published: January 2010

INTRODUCTION

This protocol describes a technique for the simultaneous monitoring of free calcium concentrations in the cytosol ([Ca2+]i) and within the lumen of the endoplasmic reticulum (ER) ([Ca2+]L) of cultured/freshly isolated dorsal root ganglia (DRG) neurons. The method uses two synthetic fluorescent Ca2+ probes (fluo-3 and mag-fura-2) in combination with fluorescence microscopy and a whole-cell patch-clamp technique. This approach has been used successfully in acutely isolated/cultured DRG neurons, in Purkinje neurons, acutely isolated from cerebellar slices, and in cultured astrocytes. In this protocol, isolated neurons are first loaded with the membrane-permeant, low-affinity Ca2+ indicator, mag-fura-2, which preferentially, though not exclusively, accumulates in the ER. Cells are then loaded with the membrane-impermeant, high-affinity calcium indicator fluo-3 using the whole-cell patch-clamp configuration. This second loading removes the majority of cytosolic mag-fura-2, replacing it with fluo-3. Mag-fura-2 and fluo-3 signals can be separated by virtue of their distinct excitation properties (340 nm and 380 nm for mag-fura-2, and 488 nm for fluo-3). An equation is provided to determine [Ca2+]L values using the 340/380 nm ratio.
Phloroglucinol Stain for Lignin
Authors: Liljegren Sarah
Published: January 2010

INTRODUCTION

The lignin found in plant cell walls can be stained with phloroglucinol in the presence of alcohol and HCl, as described in this article. The cell walls will temporarily turn red.
Rabbit Mating and Embryo Isolation
Authors: Püschel Bernd , Viebahn Christoph
Published: January 2010

INTRODUCTION

This protocol describes how to generate and collect rabbit embryos of defined developmental stages for experiments such as live embryo micromanipulation and culture or whole-mount in situ hybridization and immunofluorescence. Most values and measurements given in the procedure are optimally suited for the New Zealand White strain (the most commonly used strain because of its relatively large size and therefore large blood volumes for polyclonal antiserum collection). Successful embryo generation and isolation depend on a well-equipped animal care facility and dedicated veterinary care.
Live Rabbit Embryo Culture: Figure 1.
Authors: Püschel Bernd , Bitzer Eva , Viebahn Christoph
Published: January 2010

INTRODUCTION

This protocol outlines methods for culturing rabbit embryos. Such cultures can be used to study the impact of micromanipulation, transplantation, and the influence of signaling molecules on embryonic development. They can be performed on whole blastocysts submerged in culture medium (suspension culture) or on explanted parts of the embryo spread out on a semisolid agarose support (flat or ring culture). Cultures can be adapted to many desired growth conditions by varying the parameters, but culture length is limited to ~24 h because longer incubation times lead either to bursting of the blastocyst and subsequent rolling up of the embryonic disc or to inadequate growth, resulting in deformed embryos.
Staining and Imaging of Live Rabbit Embryos: Figure 1.
Authors: Püschel Bernd , Viebahn Christoph
Published: January 2010

INTRODUCTION

This protocol describes methods for supravital labeling of cellular proliferation and movement in rabbit embryos as well as for imaging of live cell movements. The techniques use localized labeling of epiblast cells with DiI by injection or by using comprehensive nuclear staining of all cell layers before and during early gastrulation. Because rabbit embryos implant relatively late during embryonic development, gastrulation-stage embryos can be isolated by flushing them from the uterus. Moreover, the embryonic disc is flat and translucent, providing a direct uncompromised overview of gastrulation while the whole blastocyst is protected by the zona pellucida. These properties allow examination of whole blastocysts either under the impact of changing environmental conditions (e.g., medium additives, oxygen concentrations) or following mechanical manipulation of the embryonic disc using a needle penetrating the zona pellucida. Further manipulation (e.g., transplantation) can be performed after removing the embryo from the zona and explantation of the embryonic disc. These techniques allow embryonic growth centers to be identified and single-cell movements to be recorded.
Whole-Mount In Situ Immunofluorescence of Early Rabbit Embryos
Authors: Püschel Bernd , Viebahn Christoph
Published: January 2010

INTRODUCTION

In this procedure for whole-mount immunostaining of early rabbit embryos, embryonic tissues are immersed in a small volume of buffer containing detergents to permeabilize tissues and the cells, allowing antibodies access to intracellular structures. This method allows the detection of specific cell types and the identification of subcellular characteristics. Correlating the position of the cells of interest with the stage of development allows conclusions to be drawn as to cell differentiation and embryonic development. This method can be applied to rabbit embryos from the early cleavage stages up to neurulating embryos at day 8 of embryonic development.
Artificial Insemination in Rabbits
Authors: Daniel Nathalie , Renard Jean-Paul
Published: January 2010

INTRODUCTION

Artificial insemination (AI) is used in rabbit husbandry to improve breeding management. Conception rates after AI with fresh semen are equivalent to or even better than those obtained by natural mating. Also, AI has the advantage of avoiding contact between animals. Furthermore, AI with frozen sperm is particularly useful for assisted reproduction and conservation of rabbit genetic resources, and in some cases, frozen semen produce results similar to those obtained in AI using fresh sperm. This protocol describes the most commonly used protocol for AI using fresh sperm.
Immunocytochemistry of Paramecium Cytoskeletal Structures
Authors: Beisson Janine , Bétermier Mireille , Bré Marie-Hélène , Cohen Jean , Duharcourt Sandra , Duret Laurent , Kung Ching , Malinsky Sophie , Meyer Eric , Preer John R. , Sperling Linda
Published: January 2010

INTRODUCTION

Paramecium has a highly complex cellular structure. The cytoskeleton of these organisms interacts with cilia at the cell surface to control cell movement, feeding, and environmental responses. Basal bodies anchored in the cortex organize the cytoskeleton and determine cell shape, and a conspicuous cytoskeletal network, the infraciliary lattice, underlies the cell surface and plays an important role in cellular Ca2+ homeostasis. This protocol describes methods to visualize the cytoskeleton, especially cortical structures. It is not directly applicable for visualization of membrane proteins.
Preparation and Culture of Limb Blastema Stem Cells from Regenerating Larval and Adult Salamanders
Authors: Kumar Anoop , Godwin James W.
Published: January 2010

INTRODUCTION

Adult salamanders show extensive ability to regenerate their body parts, and this can be attributed at least in part to their cellular plasticity. Cell reprogramming, dedifferentiation, proliferation and redifferentiation occur naturally in adult salamanders in response to tissue damage or removal. Limb regeneration proceeds by mesenchymal dedifferentiation and cell migration at the end of the stump. This generates a cohort of mesenchymal stem cells called blastema cells, which accumulate under the wound epithelium. The “limb blastema” is an autonomous structure that retains the positional identity of its location of origin as well as morphogenetic cues for reconstructing a new limb. At the cellular level, blastema stem cells maintain characteristics such as positional memory derived from their location, while expressing new markers relevant to the process of limb regeneration. In this protocol, a nonenzymatic method is described for the dissociation of blastema stem cells from newts (Notophthalmus viridescens) and axolotls (Ambystoma mexicanum).
Using the Metagenomics RAST Server (MG-RAST) for Analyzing Shotgun Metagenomes
Authors: Glass Elizabeth M. , Wilkening Jared , Wilke Andreas , Antonopoulos Dionysios , Meyer Folker
Published: January 2010

INTRODUCTION

Shotgun metagenomics creates millions of fragments of short DNA reads, which are meaningless unless analyzed appropriately. The Metagenomics RAST server (MG-RAST) is a web-based, open source system that offers a unique suite of tools for analyzing these data sets. After de-replication and quality control, fragments are mapped against a comprehensive nonredundant database (NR). Phylogenetic and metabolic reconstructions are computed from the set of hits against the NR. The resulting data are made available for browsing, download, and most importantly, comparison against a comprehensive collection of public metagenomes. A submitted metagenome is visible only to the user, unless the user makes it public or shares with other registered users. Public metagenomes are available to all.
Dual-Color ELISPOT Assay for the Simultaneous Detection of IL-2 and/or IFN-γ Secreting T Cells
Authors: Boulet Salix , Ndongala Michel L. , Bernard Nicole F.
Published: January 2010

INTRODUCTION

The enzyme-linked immunospot (ELISPOT) assay measures the secretion intensity of effector molecules released by immune cells in response to ex vivo antigenic stimulation, as well as the frequency of these responding cells. This assay is highly sensitive, quantitative, easy to use, and amenable to high-throughput screening. For these reasons, the ELISPOT assay is considered by many as a gold standard for monitoring cellular immune responses. Until recently, ELISPOT assays using chromophores to detect the T cell secretion of cytokines were limited to the characterization of a single effector molecule. Notably, studies evaluating the immune response to chronic viral infections often measured IFN-γ secretion by ELISPOT because of the known antiviral effects of this cytokine as well as its correlation to the cytotoxic capacity of T cells. However, maintenance of both IFN-γ and IL-2 secretion by pathogen-specific T cells has been linked to a more favorable clinical outcome in human immunodeficiency virus (HIV) and Leishmania infections. Therefore, an ELISPOT assay able to simultaneously characterize T cell responses in terms of IL-2 and IFN-γ secretion is potentially relevant for the monitoring of immune responses to certain infectious agents. In this protocol, we describe an ELISPOT assay for the simultaneous detection of IL-2 and IFN-γ upon stimulation with viral peptides.
The conflation of ignorance and knowledge in the inference of clade posteriors
Authors: Randle Christopher P. , Pickett Kurt M.
Published: January 2010

The objective Bayesian approach relies on the construction of prior distributions that reflect ignorance. When topologies are considered equally probablea priori, clades cannot be. Shifting justifications have been offered for the use of uniform topological priors in Bayesian inference. These include: (i) topological priors do not inappropriately influence Bayesian inference when they are uniform; (ii) although clade priors are not uniform, their undesirable influence is negated by the likelihood function, even when data sets are small; and (iii) the influence of nonuniform clade priors is an appropriate reflection of knowledge. The first two justifications have been addressed previously: the first is false, and the second was found to be questionable. The third and most recent justification is inconsistent with the first two, and with the objective Bayesian philosophy itself. Thus, there has been no coherent justification for the use of nonflat clade priors in Bayesian phylogenetics. We discuss several solutions: (i) Bayesian inference can be abandoned in favour of other methods of phylogenetic inference; (ii) the objective Bayesian philosophy can be abandoned in favour of a subjective interpretation; (iii) the topology with the greatest posterior probability, which is also the tree of greatest marginal likelihood, can be accepted as optimal, with clade support estimated using other means; or (iv) a Bayes factor, which accounts for differences in priors among competing hypotheses, can be used to assess the weight of evidence in support of clades. ©The Willi Hennig Society 2009
Population size of lions in Yankari Game Reserve as revealed by faecal DNA sampling
Authors: Tende Talatu , Ottosson Ulf , Hansson Bengt , Åkesson Mikael , Bensch Staffan
Published: January 2010

Studies have shown that lion (Panthera leo) populations in West Africa are small, isolated and fragmented. In Nigeria, lions have disappeared from unprotected areas and are nowadays found only in parks and reserves where these populations may still decline. It is therefore urgent to obtain reliable estimates of population sizes at different localities. Direct observational surveys may either fail to count all individuals or count some individuals repeatedly and are therefore associated with unknown levels of estimation errors. More accurate estimates can be obtained if direct counting is combined with DNA‐based individual identification. As lions are difficult to identify individually, presented here is a method that can be a valuable addition to the existing census methods.
Systematic review: the effects of carbonated beverages on gastro‐oesophageal reflux disease
Authors: JOHNSON T. , GERSON L. , HERSHCOVICI T. , STAVE C. , FASS R.
Published: January 2010

Aliment Pharmacol Ther 31, 607–614

Background  Carbonated beverages have unique properties that may potentially exacerbate gastro‐oesophageal reflux disease (GERD), such as high acidity and carbonation. Cessation of carbonated beverage consumption is commonly recommended as part of lifestyle modifications for patients with GERD.

Aims  To evaluate the relationship of carbonated beverages with oesophageal pH, oesophageal motility, oesophageal damage, GERD symptoms and GERD complications.

Methods  A systematic review.

Results  Carbonated beverage consumption results in a very short decline in intra‐oesophageal pH. In addition, carbonated beverages may lead to a transient reduction in lower oesophageal sphincter basal pressure. There is no evidence that carbonated beverages directly cause oesophageal damage. Carbonated beverages have not been consistently shown to cause GERD‐related symptoms. Furthermore, there is no evidence that these popular drinks lead to GERD complications or oesophageal cancer.

Conclusions  Based on the currently available literature, it appears that there is no direct evidence that carbonated beverages promote or exacerbate GERD.
Does strong selection promote host specialisation and ecological speciation in insect herbivores? Evidence from Neochlamisus leaf beetles
Authors: FUNK DANIEL J.
Published: January 2010

1. Outstanding issues in evolutionary ecology include: (1) the prevalence of host‐associated ecological specialisation among herbivorous insects, and (2) the speciation processes contributing to the tremendous species‐level diversity of herbivores.

2. Neochlamisus leaf beetle species are generally monophagous, but cumulatively employ a disparate array of host plants. These patterns occur in microcosm within the putatively oligophagous Neochlamisus bebbianae, which uses specific host species within each of six plant genera representing five families. I refer to all beetle populations associated with any one of these hosts as a particular ‘host form’.

3. This paper describes aspects of Neochlamisus biology and some experimental findings relevant to both host‐use evolution and host specialisation in Neochlamisus, and to ecological divergence, reproductive isolation, and genetic differentiation among N. bebbianae host forms. It thereby reviews a research programme focused on ecological speciation.

4. With respect to these host forms, our findings include: (1) ubiquitous host specialisation, (2) constraints on ‘multitasking’ that select for host specialisation, (3) ubiquitous ecological divergence, (4) pre‐ and post‐mating reproductive barriers, (5) genetic and environmental contributions to ecological divergence and reproductive isolation, (6) positive statistical associations between ecological divergence and reproductive isolation, (7) strongly differentiated loci despite gene flow, reflecting divergent host‐related selection, and (8) possible host race formation. We additionally describe a unique example of intimate host adaptation in another species, Neochlamisus platani.

5. The intimate and discrete nature of herbivore–host associations may generate unusually strong (host‐related) selection that could simultaneous explain pronounced rates of herbivore specialisation and speciation.
DNA profiling of host–herbivore interactions in tropical forests
Authors: NAVARRO SARA PINZÓN , JURADO‐RIVERA JOSÉ A. , GÓMEZ‐ZURITA JESÚS , LYAL CHRISTOPHER H. C. , VOGLER ALFRIED P.
Published: January 2010

1. The diversity of insects in tropical forests remains poorly known, in particular regarding the critical feeding associations of herbivores, which are thought to drive species richness in these ecosystems.

2. Host records remain elusive and traditionally require labour‐intensive feeding trials. A recent approach analyses plant DNA ingested by herbivorous insects; direct PCR amplification from DNA extracts from weevils (Curculionoidea) using chloroplast (trnL intron) primers was successful in 41 of 115 cases, resulting in 40 different sequences.

3. The resulting trnL intron sequences were identified against public databases to various hierarchical levels based on their position in phylogenetic trees and shown to be members of 26 plant families from different major groups of angiosperms.

4. Among the trnL intron sequences, seven pairs or triplets of close relatives (0–2 bp difference) were found which may represent intraspecific variation in the respective host plants.

5. Molecular clock calibrations of mitochondrial cox1 sequences of weevils established great distances of lineages obtained (all splits estimated >20 Mya). Distant taxa were found to feed on the same or similar hosts in some cases, showing low evolutionary conservation of host associations among deeper levels.

6. The technique provides a new means of studying species diversity and plant–herbivore interactions in tropical forests, and removes the constraints of the need for actual observations of feeding in ecological and evolutionary studies.
The pea aphid complex as a model of ecological speciation
Authors: JEAN PECCOUD , JEAN‐CHRISTOPHE SIMON
Published: January 2010

1. Host‐specialised races of plant‐feeding insects are particularly informative models in the study of ecological speciation, that is, the evolution of reproductive isolation through divergent natural selection. However, within the enormous diversity of phytophagous insects, the mechanisms of ecological divergence have been elucidated in few host race systems.

2. Here we review the literature covering speciation through host‐plant specialisation in a well‐studied model, the pea aphid complex,Acyrthosiphon pisum, which encompasses numerous biotypes that parasitise different legume host species worldwide.

3. Published results are consistent with ecologically promoted reproductive isolation. Divergent host‐induced selection is pronounced across biotypes, and reflects genetic trade‐offs preventing the optimal use of multiple host plants. While these genetic trade‐offs may partly explain the unfitness of hybrids between biotypes, hybridisation occurring on plants is also limited by genetically‐based host preference, and by selection against migrants that chose unfavourable hosts. The continuum of genetic divergence displayed by 11 races and species of the pea aphid complex suggests that host races constitute an intermediate step in the speciation process, and that host specialisation may indeed lead to complete speciation. Uncertainties remain on the contribution of non‐ecological reproductive barriers to biotype divergence and on the physiological and molecular bases of host specialisation.
Speciation in fig wasps
Authors: COOK JAMES M. , SEGAR SIMON T.
Published: January 2010

1. There are over 700 species of fig trees in the tropics and several thousand species of fig wasps are associated with their syconia (inflorescences). These wasps comprise a monophyletic family of fig pollinators and several diverse lineages of non‐pollinating wasps. The pollinator larvae gall fig flowers, while larvae of non‐pollinating species either initiate their own galls or parasitise the galls of other wasps.

2. A single fig species has one to four pollinator species and also hosts up to 30 non‐pollinating wasp species. Most wasps show a high degree of host‐plant specificity and are known from only a single fig species. However, in some cases wasps may be shared across closely related fig species.

3. There is impressive morphological co‐evolution between figs and fig wasps and this, combined with a high degree of partner specificity, led to the expectation that figs and pollinators have cospeciated extensively. Comparison of deep phylogenies supports long‐term codivergence of figs and pollinators, but also suggests that some host shifts have occurred.

4. Phylogenies of more closely related species do not match perfectly and may even be incongruent, suggesting significant roles for processes other than strict cospeciation. Combined with recent evidence on host specificity patterns, this suggests that pollinator wasps may often speciate by host shifts between closely related figs, or by duplication (the wasp speciates but the fig doesn't). The frequencies and biological details of these different modes of speciation invite further study.

5. Far less is known about speciation in non‐pollinating fig wasps. Some lineages have probably co‐evolved with figs and pollinators for most of the evolutionary history of the symbiosis, while others appear to be more recent colonisers. Many species appear to be highly host‐plant specific, but those that lay eggs through the fig wall without entering the syconium (the majority of species) may be subject to fewer constraints on host shifting than pollinators. There is evidence for substantial host shifting in at least one genus, but also evidence for ecological speciation on the same host plant by niche shifts in other cases.

6. Finally, recent work has begun to address the issue of ‘community phylogeny’ and provided evidence for long‐term co‐divergence of multiple pollinating and non‐pollinating wasp lineages with their host figs.
Evolutionary aspects of ecological generalism with special reference to conservation biological control
Authors: EMELIANOV IGOR
Published: January 2010

1. If ecological generalism is an evolving heritable trait, we need to study the underlying genetics in order to understand the nature of this fundamental phenomenon.

2. I consider two alternatives. Under the Type I mechanism, ecological generalism is exclusively a population property. Under the Type II mechanism, generalism can vary among genotypes as well as among populations.

3. The genetics underlying the Type I mechanism is straightforward, whereas Type II genetics is less so. I propose that a broad adaptive range of generalist genotypes can be a result of a pleiotropic modifier that, when expressed, renders fitness of the carrier relatively insensitive to environmental variance.

4. A review of the theory and the data indicates that stable ecological generalism is more likely to occur under the Type II compared to the Type I mechanism. Type II generalism appears to be relatively more common and more compatible with patterns observed in the wild.

5. Generalism of natural enemies is a key factor influencing the effectiveness of conservation biological control (CBC). I argue that natural enemies with Type II generalism are relatively more suitable for CBC applications, and spell out directions for further research.
Venous access in haemophilic children: choice and management
Authors: SANTAGOSTINO E. , MANCUSO M. E.
Published: January 2010

Summary.  Current haemophilia treatment in children is based on regular intravenous infusions of concentrates for prolonged periods, according to prophylaxis regimens or immune tolerance induction treatment, in cases of inhibitor development. Therefore, a stable and uncomplicated venous access is required and as such peripheral veins represent the preferred option. However, frequent infusions in the home setting can be problematic in very young children and for this reason, central venous access devices (CVADs) have been widely used to improve treatment feasibility. Unfortunately CVADs’ use is associated with a high rate of complications, and infections and thrombotic occlusion can influence treatment outcome by causing unwanted treatment interruption. CVADs can be grouped into three main categories: external non‐tunnelled, external tunnelled and fully implantable devices known as ports. The management of CVADs at home often represents a challenge because a strict adherence to sterile procedures is required. Indeed, the incidence of infections with ports is much lower than that reported for external devices; however, ports carry the inconvenience of needle sticks. More recently, arteriovenous fistula was shown to be a suitable alternative to CVADs because it is easy to use and is associated with a lower rate of complication.