bluuebunny/crossref_metadata_2025 huggingface dataset:
The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Authors: Sakurai-Yageta Mika , Recchi Chiara , Le Dez Gaëlle , Sibarita Jean-Baptiste , Daviet Laurent , Camonis Jacques , D'Souza-Schorey Crislyn , Chavrier Philippe
Published: June 2008

Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.
CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation
Authors: Li Xueni , Huang Mei , Zheng Huiling , Wang Yinyin , Ren Fangli , Shang Yu , Zhai Yonggong , Irwin David M. , Shi Yuguang , Chen Di , Chang Zhijie
Published: June 2008

Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage.
Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores
Authors: Tighe Anthony , Staples Oliver , Taylor Stephen
Published: June 2008

Mps1 is an upstream component of the spindle assembly checkpoint, which, in human cells, is required for checkpoint activation in response to spindle damage but not apparently during an unperturbed mitosis. Mps1 also recruits Mad1 and Mad2 to kinetochores. However, whether the enzymatic activity of Mps1 is required for these processes is unclear. To address this question, we established an RNA interference (RNAi) complementation assay. Repression of Mps1 triggers premature anaphase, often with unaligned or maloriented chromosomes. This phenotype is rescued by an RNAi-resistant wild-type Mps1 transgene but not by a catalytically inactive mutant. An analogue-sensitive allele, Mps1M602A, also rescues the RNAi-induced defect, but not when inhibited by the adenosine triphosphate analogue 1-NM-PP1. Thus, Mps1 activity does restrain anaphase during an unperturbed mitosis. Furthermore, although catalytically inactive Mps1 can restore kinetochore localization of Mad1, only the active kinase restores Mad2 localization. Thus, in human cells, Mps1 catalytic activity is required for spindle checkpoint function and recruitment of Mad2.
Sγ3 switch sequences function in place of endogenous Sγ1 to mediate antibody class switching
Authors: Zarrin Ali A. , Goff Peter H. , Senger Kate , Alt Frederick W.
Published: June 2008

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cμ exons with a set of downstream IgH constant region (CH) exons. Individual sets of CH exons are flanked upstream by long (1–10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Sμ region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region–specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sγ1 region was replaced with wild-type (WT) or synthetic 2-kb Sγ3 sequences or a synthetic 2-kb Sγ1 sequence. We found that both the inserted endogenous and synthetic Sγ3 sequences functioned similarly to a size-matched synthetic Sγ1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sγ3 can function similarly to Sγ1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype–specific functions of other endogenous S regions.
An MHC class Ib–restricted CD8 T cell response confers antiviral immunity
Authors: Swanson Phillip A. , Pack Christopher D. , Hadley Annette , Wang Chyung-Ru , Stroynowski Iwona , Jensen Peter E. , Lukacher Aron E.
Published: June 2008

Although immunity against intracellular pathogens is primarily provided by CD8 T lymphocytes that recognize pathogen-derived peptides presented by major histocompatibility complex (MHC) class Ia molecules, MHC class Ib–restricted CD8 T cells have been implicated in antiviral immunity. Using mouse polyoma virus (PyV), we found that MHC class Ia–deficient (Kb−/−Db−/−) mice efficiently control this persistently infecting mouse pathogen. CD8 T cell depletion mitigates clearance of PyV in Kb−/−Db−/− mice. We identified the ligand for PyV-specific CD8 T cells in Kb−/−Db−/− mice as a nonamer peptide from the VP2 capsid protein presented by Q9, a member of the β2 microglobulin–associated Qa-2 family. Using Q9-VP2 tetramers, we monitored delayed but progressive expansion of these antigen-specific CD8αβ T cells in Kb−/−Db−/− mice. Importantly, we demonstrate that Q9-VP2–specific CD8 T cells more effectively clear wild-type PyV than a VP2 epitopenull mutant PyV. Finally, we show that wild-type mice also generate Q9-restricted VP2 epitope–specific CD8 T cells to PyV infection. To our knowledge, this is the first evidence for a defined MHC class Ib–restricted antiviral CD8 T cell response that contributes to host defense. This study motivates efforts to uncover MHC class Ib–restricted CD8 T cell responses in other viral infections, and given the limited polymorphism of MHC class Ib molecules, it raises the possibility of developing peptide-based viral vaccines having broad coverage across MHC haplotypes.
A New Solution to the Puzzle of Simplicity
Authors: Kelly Kevin T.
Published: June 2008

Explaining the connection, if any, between simplicity and truth is among the deepest problems facing the philosophy of science, statistics, and machine learning. Say that an efficient truth finding method minimizes worst case costs en route to converging to the true answer to a theory choice problem. Let the costs considered include the number of times a false answer is selected, the number of times opinion is reversed, and the times at which the reversals occur. It is demonstrated that (1) always choosing the simplest theory compatible with experience, and (2) hanging onto it while it remains simplest, is both necessary and sufficient for efficiency.
The Communication Structure of Epistemic Communities
Authors: Zollman Kevin J. S.
Published: June 2008

Increasingly, epistemologists are becoming interested in social structures and their effect on epistemic enterprises, but little attention has been paid to the proper distribution of experimental results among scientists. This paper will analyze a model first suggested by two economists, which nicely captures one type of learning situation faced by scientists. The results of a computer simulation study of this model provide two interesting conclusions. First, in some contexts, a community of scientists is, as a whole, more reliable when its members are less aware of their colleagues’ experimental results. Second, there is a robust tradeoff between the reliability of a community and the speed with which it reaches a correct conclusion.
A Philosopher's Guide to Empirical Success
Authors: Forster Malcolm R.
Published: June 2008

The simple question, what is empirical success? turns out to have a surprisingly complicated answer. We need to distinguish between meritorious fit and ‘fudged fit’, which is akin to the distinction between prediction and accommodation. The final proposal is that empirical success emerges in a theory dependent way from the agreement of independent measurements of theoretically postulated quantities. Implications for realism and Bayesianism are discussed.
Neo-functional Analysis: Phylogenetical Restrictions on Causal Role Functions
Authors: Šustar Predrag
Published: June 2008

The most recent resurgence of philosophical attention to the so-called ‘functional talk’ in the sciences can be summarized in terms of the following questions: (Q1) What kind ofrestrictions, and in particular, what kind ofevolutionary restrictionsas well as to what extent, is involved in functional ascriptions? (Q2) How can we account for theexplanatory importof function-ascribing statements? This paper addresses these questions through a modified version of Cummins’ functional analysis. The modification in question is concerned with phylogenetical restrictions on causal role functions, and it stems from an analysis of some primary areas in molecular biology. I examine how evolutionary consideration affects the so-called ‘function-analytical explanatory strategy’ (Cummins [1975] 1998, 2002). Finally, I argue that the neo-functional analysis here proposed accounts for a certain convergence between the main rival theories of biological function.
Seeing the Forest and the Trees: Realism about Communities and Ecosystems
Authors: Odenbaugh Jay
Published: June 2008

In this essay I first provide an analysis of various community concepts. Second, I evaluate two of the most serious challenges to the existence of communities—gradient and paleoecological analysis respectively—arguing that, properly understood, neither threatens the existence of communities construed interactively. Finally, I apply the same interactive approach to ecosystem ecology, arguing that ecosystems may exist robustly as well.
The Empirical Inadequacy of Species Cohesion by Gene Flow
Authors: Barker Matthew J.
Published: June 2008

This paper brings needed clarity to the influential view that species are cohesive entities held together by gene flow, and then develops an empirical argument against that view: Neglected data suggest gene flow is neither necessary nor sufficient for species cohesion. Implications are discussed.
Putting Expectations in Order
Authors: Baker Alan
Published: June 2008

In their paper, “Vexing Expectations,” Nover and Hájek (2004) present an allegedly paradoxical betting scenario which they call the Pasadena Game (PG). They argue that the silence of standard decision theory concerning the value of playing PG poses a serious problem. This paper provides a threefold response. First, I argue that the real problem is not that decision theory is “silent” concerning PG, but that it delivers multiple conflicting verdicts. Second, I offer a diagnosis of the problem based on the insight that standard decision theory is, rightly, sensitive to order. Third, I describe a new betting scenario—the Alternating St. Petersburg Game—which is genuinely paradoxical. Standard decision theory is silent on the value of playing this game even if restrictions are placed on the order in which the various alternative payoffs are summed.
Does Evidential Variety Depend on How the Evidence Is Described?
Authors: Novack Greg
Published: June 2008

The Variety of Evidence Thesis (VET) says that (ceteris paribus) the more diverse (or varied) of two bodies of evidence is the more confirmatory of a hypothesis H. Two recent types of Bayesian explication of VET account for the intuitive force of VET by defining variety as some function of the probabilities of the propositions which collectively constitute a body of evidence. I show that these two accounts of VET are not tracking a meaningful property of bodies of evidence, but rather are tracking artifacts of how those bodies of evidence are described. According to each account, whether a body of evidence is more varied than another depends on how the bodies are split into parts. Furthermore, for each type of account there exists a way to redescribe the total evidence such that any two totalities are equally varied.
Dependency Equilibria
Authors: Spohn Wolfgang
Published: June 2008

This paper introduces a new equilibrium concept for normal form games called dependency equilibrium; it is defined, exemplified, and compared with Nash and correlated equilibria in Sections 2–4. Its philosophical motive is to rationalize cooperation in the one shot prisoners’ dilemma. A brief discussion of its meaningfulness in Section 5 concludes the paper.
Optimal Judgment Aggregation
Authors: Bonilla Jesús Zamora
Published: June 2008

The constitution of a collective judgment is analyzed from a contractarian point of view. The optimal collective judgment is defined as the one that maximizes the sum of the utility each member gets from the collective adoption of that judgment. It is argued that judgment aggregation is a different process from the aggregation of information and public deliberation. This entails that the adoption of a collective judgment should not make any rational member of the group change her individual opinion, and so the collective judgment can not have any kind of epistemic superiority over the individual ones.
Robustness in Signaling Games
Authors: Huttegger Simon M.
Published: June 2008

The spontaneous emergence of signaling has already been studied in terms of standard evolutionary dynamics of signaling games. Standard evolutionary dynamics is given by the replicator equations. Thus, it is not clear whether the results for standard evolutionary dynamics depend crucially on the functional form of the replicator equations. In this paper I show that the basic results for the replicator dynamics of signaling games carry over to a number of other evolutionary dynamics.
Robust Simulations
Authors: Muldoon Ryan
Published: June 2008

As scientists begin to study increasingly complex questions, many have turned to computer simulation to assist in their inquiry. This methodology has been challenged by both analytic modelers and experimentalists. A primary objection of analytic modelers is that simulations are simply too complicated to perform model verification. From the experimentalist perspective it is that there is no means to demonstrate the reality of simulation. The aim of this paper is to consider objections from both of these perspectives, and to argue that a proper understanding and application of robustness analysis is able to resolve them.
Model Selection and the Multiplicity of Patterns in Empirical Data
Authors: McAllister James W.
Published: June 2008

Several quantitative techniques for choosing among data models are available. Among these are techniques based on algorithmic information theory, minimum description length theory, and the Akaike information criterion. All these techniques are designed to identify a single model of a data set as being the closest to the truth. I argue, using examples, that many data sets in science show multiple patterns, providing evidence for multiple phenomena. For any such data set, there is more than one data model that must be considered close to the truth. I conclude that, since the established techniques for choosing among data models are unequipped to handle these cases, they cannot be regarded as adequate.
Indeterminism, Asymptotic Reasoning, and Time Irreversibility in Classical Physics
Authors: Korolev Alexandre
Published: June 2008

A recent proposal by Norton (2003) to show that a simple Newtonian system can exhibit stochastic acausal behavior by giving rise to spontaneous movements of a mass on the dome of a certain shape is examined. We discuss the physical significance of an often overlooked and yet important Lipschitz condition the violation of which leads to the existence of anomalous nontrivial solutions in this and similar cases. We show that the Lipschitz condition is closely linked with the time reversibility of certain solutions in Newtonian mechanics and the failure to incorporate this condition within Newtonian mechanics may unsurprisingly lead to physically impossible solutions that have no serious metaphysical implications.
A General Conceptual Framework for Decoherence in Closed and Open Systems
Authors: Castagnino Mario , Laura Roberto , Lombardi Olimpia
Published: June 2008

In this paper we argue that the formalisms for decoherence originally devised to deal just with closed or open systems can be subsumed under a general conceptual framework, in such a way that they cooperate in the understanding of the same physical phenomenon. This new perspective dissolves certain conceptual difficulties of the einselection program but, at the same time, shows that the openness of the quantum system is not the essential ingredient for decoherence.
Interventions and Causal Inference
Authors: Eberhardt Frederick , Scheines Richard
Published: June 2008

The literature on causal discovery has focused on interventions that involve randomly assigning values to a single variable. But such a randomized intervention is not the only possibility, nor is it always optimal. In some cases it is impossible or it would be unethical to perform such an intervention. We provide an account of ‘hard’ and ‘soft’ interventions and discuss what they can contribute to causal discovery. We also describe how the choice of the optimal intervention(s) depends heavily on the particular experimental setup and the assumptions that can be made.
Is Measurement a Black Box? On the Importance of Understanding Measurement Even in Quantum Information and Computation
Authors: Dickson Michael
Published: June 2008

It has been argued, partly from the lack of any widely accepted solution to the measurement problem, and partly from recent results from quantum information theory, that measurement in quantum theory is best treated as a black box. However, there is a crucial difference between ‘having no account of measurement’ and ‘having no solution to the measurement problem’. We know a lot about measurements. Taking into account this knowledge sheds light on quantum theory as a theory of information and computation. In particular, the scheme of ‘one-way quantnum computation’ takes on a new character in light of the role that reference frames play in actually carrying out any one-way quantum comptuation.
Why Functional Form Matters: Revealing the Structure in Structural Models in Econometrics
Authors: Fennell Damien
Published: June 2008

This paper argues that econometricians’ explicit adoption of identification conditions in structural equation modelling commits them to read the functional form of their equations in a strong, nonmathematical way. This content, which is implicitly attributed to the functional form of structural equations, is part of what makes equation structural. Unfortunately, econometricians are not explicit about the role functional form plays in signifying structural content. In order to remedy this, the second part of this paper presents an interpretation of the functional form based on Herbert Simon's definition of causal order. This begins to set out just what the functional form of structural equations represents.
The conversion of the thermal energy of plasma in the SOL of tokamaks
Authors: Nedospasov A.V. , Nenova N.V.
Published: June 2008

When the plasma expands across the confining magnetic field, a part of its thermal energy is converted to electrical energy. In the SOL of tokamaks, the motion of the plasma across the field due to turbulent processes is accompanied by its departure along the open lines of the magnetic field. The conversion of thermal energy is taken into account in theoretical studies devoted to the physics of plasma in the SOL; however, this conversion is ignored in numerical models, for example, in B2–SOLPS4.0.

This paper deals with thermal-to-electrical energy conversion in the SOL of tokamaks. It is demonstrated that the part of the thermal energy subjected to conversion to electrical energy forms an appreciable part of the total energy flowing in the SOL. In ITER, this fraction may be as high as 20–25%. The electrical energy generated in the SOL volume is liberated in the form of Joule heat in a relatively cold plasma in the vicinity of diverter plates or directly on these plates.
An empirical scaling for deuterium retention in co-deposited beryllium layers
Authors: De Temmerman G. , Baldwin M.J. , Doerner R.P. , Nishijima D. , Schmid K.
Published: June 2008

Different mechanisms contribute to tritium retention in ITER, amongst which co-deposition with materials from the plasma-facing components is one of the main contributors. A systematic study of the influence of the deposition conditions (substrate temperature, deposition rate, energy of the incident particles) on the deuterium retention in co-deposited beryllium layers has been carried out in PISCES-B. The mechanism by which deuterium co-deposits with beryllium appears to be a combination of co-deposition and implantation, with a decreased retention for increased deposition rate and an increased retention for increased incident deuterium particle energy. A scaling equation is developed, providing a method to predict the retention in Be co-deposits formed in PISCES-B as a function of the layer formation conditions. Using this equation, previously published data on retention in Be co-deposits are re-examined and relatively good agreement is found with the prediction of the scaling equation.
Genetic diversity and effective size of the Atlantic salmon Salmo salar L. inhabiting the River Eo (Spain) following a stock collapse
Authors: Ribeiro Â. , Morán P. , Caballero A.
Published: June 2008

Historic angling records suggest the occurrence of a drastic decline in the River Eo (Spain) Atlantic salmon population size during the past two decades, as a result of overexploitation and habitat deterioration. In recent years, the population has been apparently recovering, and the present study is aimed to report information on the level of genetic diversity and the effective size of the current population as these may have immediate consequences for its conservation. Eighty‐six salmon from two temporal groups (1998–1999 and 2004–2006), representing three generations, were genotyped using a panel of eight microsatellites. Inspite of the recent decline in census numbers and the detection of the signs of a population bottleneck, the population exhibits a high level of genetic diversity, similar to that from other populations, and almost unchanged during the period of study [average allelic richness (A) = 14·0 and 13·9, and average heterozygosity (He) = 0·843 and 0·851 in 1998–1999 and 2004–2006, respectively]. The effective population size (Ne) estimated by two different temporal methods showed a consistent value around 80 salmon, whereas the estimates from the linkage disequilibrium (LD) method provided a value around 165 individuals for either sample. The recent growing number of salmon, as indicated by fisheries records, the relatively large estimates of the ratio Ne/N (with range 0·23–0·44 for the temporal estimates and 0·31–0·59 for the LD estimates) and the high levels of diversity found suggest that the population has not been greatly affected by the historical census declines and can be expected to recover in the future.
Recent chromosome diversification in the evolutionary radiation of the freshwater fish family Curimatidae (Characiformes)
Authors: Venere P. C. , Souza I. L. , Silva L. K. S. , Dos Anjos M. B. , De Oliveira R. R. , Galetti P. M.
Published: June 2008

Neotropical Curimatidae fishes include 97 species in eight genera. Basic cytogenetic studies show a karyotype of 2n = 54 chromosomes in most species. Karyotype divergence of the nucleolus organizing regions between species has been reported, and these regions appear to be good cytotaxonomic markers. In the present work, karyotype, heterochromatin and Ag‐NOR variability in 13 species were investigated to analyse the chromosome diversification in view of the biogeographic history of this group. Only Cyphocharax platanus showed a karyotype with 2n = 58 chromosomes. Ag‐NOR and C‐banding patterns were quite divergent among the species studied. All species whose C‐bands were analysed had heterochromatic blocks associated with the nucleolus organizing regions. Species with multiple Ag‐NORs also showed an increase in NOR‐associated heterochromatic blocks. C‐banding showed considerable differentiation among species, revealing a pronounced chromosome diversification in this group. Karyotypic variability corroborates the hypothesis that these fishes in Amazon region show various discrete patterns of species endemism. Chromosome diversification in curimatids has a recent origin and appears to be accompanying the post‐Andean speciation responsible for the diversity of species in the family.
RNAi‐mediated gene silencing in fishes?
Authors: Schyth B. D.
Published: June 2008

The recent discovery that small regulatory RNAs have diverse roles in eukaryotic intracellular life, such as fine tuners of endogenous gene expression, as inhibitors of viral replication and as suppressors of transposon activity, is now widely recognized as a major breakthrough in the understanding of cellular biology. The pathways used by these small RNAs seem to build upon a few central mechanisms in which small double‐stranded RNAs (dsRNAs) are generated from various precursor RNAs of exogenous or endogenous origin and used for inhibiting expression of genes with sequence complementarity to the small RNA. In particular, one class of small gene‐regulatory RNAs, known as small interfering RNAs (siRNAs), has the potential for highly specific gene silencing. These are c. 21 nucleotide perfectly complementary dsRNAs with 3′ end overhangs. They are produced by cleavage of long dsRNA believed to be of viral origin. Subsequently, a large cytoplasmic protein complex known as the RNA‐induced silencing complex (RISC) uses the antisense strand of the siRNAs for targeting complementary mRNA strands followed by cleavage of these transcripts in the mechanism called RNA interference (RNAi). Accordingly, synthetic siRNAs may have a potential for treating diseases caused by intracellular pathogens like viruses by specifically targeting the expression of their molecular components. Furthermore, as target sequences need not be of viral origin, siRNAs hold a promise for the treatment of various cellular diseases and as a tool in reverse genetic studies. SiRNAs are currently exploited for such purposes in diverse organisms ranging from plants and invertebrates to human beings. But due to initial difficulties, when using RNAi for gene silencing in fishes, only a few such studies have been conducted in fishes. The purpose of this review is to inspire further experimental work.
B chromosomes: an update about their occurrence in freshwater Neotropical fishes (Teleostei)
Authors: Carvalho R. A. , Martins‐Santos I. C. , Dias A. L.
Published: June 2008

In freshwater Neotropical fishes, supernumerary chromosomes or B chromosomes have been found in 61 species so far, distributed in 16 families of seven different orders and in distinct hydrographic basins. The order Characiformes is the one that possesses the most species with these chromosomes, including 31 species from six different families. The order Siluriformes has 21 species from five families, with Rhamdia, of the family Heptapteridae, being the most common genus where these chromosomes are found. The order Perciformes has five species with B chromosomes in only one family, Cichlidae, and the orders Beloniformes, Cyprinodontiformes, Gymnotiformes and Synbranchiformes contain only one species each with extra chromosomes. They are found as microchromosomes in Schizodon, Astyanax, Moenkhausia, Cyphocharax, Steindachnerina, Prochilodus, Rhamdia, Iheringichthys and Loricaria, among others, up to supernumerary macrochromosomes as in Astyanax, varying in morphology as well as number, showing evidence of the wide diversity of B chromosomes in Neotropical fishes.
Population structure and phylogeography of Aphyocypris kikuchii (Oshima) based on mitochondrial DNA variation
Authors: Lin H.‐D. , Hsu K.‐C. , Shao K.‐T. , Chang Y.‐C. , Wang J.‐P. , Lin C.‐J. , Chiang T.‐Y.
Published: June 2008

Aphyocypris kikuchii is a cyprinid species endemic to northern and eastern Taiwan and is the only primary freshwater fish native east of the Coastal Mountain Range. In total, 92 individuals of A. kikuchii from seven populations in three regions of the island were surveyed for mitochondrial DNA (mtDNA) variation. High haplotype diversity (h= 0·989) and low nucleotide diversity (π= 0·009) of mtDNA were detected. Negative values of Tajima’s D and unimodal mismatch distributions probably reflect a history of recent demographic expansions from small populations. Three major haplotype clusters displayed geographically non‐overlapping distributions, indicating a long‐term isolation between regions. Hierarchical analysis of molecular variance showed significant genetic structuring among populations (ΦST= 0·66). Significant haplotype heterogeneity was also detected among populations within regions (ΦSC= 0·41, P < 0·001) and among regions (ΦCT= 0·43, P < 0·05). Molecular clock estimates of coalescence in the three major mtDNA lineages indicated coalescence in the most recent common ancestor c. 0·11–0·39 million years ago. Haplotypes of cluster B nested as interior nodes in the haplotype network, indicating that migrations from Shueilian (SL) populations to the northern region (cluster A) and to the eastern region (cluster C) may have occurred independently. Lineages A and B + C should be managed as two distinct evolutionarily significant units, while the northern, SL and southern groups should be managed as separate management units.
A new epithelial‐like cell line from eye muscle of catla Catla catla (Hamilton): development and characterization
Authors: Ishaq Ahmed V. P. , Chandra V. , Parameswaran V. , Venkatesan C. , Shukla R. , Bhonde R. , Sahul Hameed A. S.
Published: June 2008

A new cell line [Sahul India Catla Eye (SICE)] has been developed from eye tissue of Indian major carp (Catla catla), a freshwater fish cultivated in India. The cell line was maintained in Leibovitz’s L‐15 supplemented with 15% foetal bovine serum (FBS). These cells have been subcultured >80 times over a period of 1·5 years. This cell line has been designated SICE. The SICE cell line consists predominantly of epithelial‐like cells. These cells are strongly positive for epithelial markers such as pancytokeratin and cytokeratin 19. The cells were able to grow at temperatures between 25 and 32° C with optimum temperature of 28° C. The growth rate of catla eye cells increased as the FBS proportion increased from 2 to 20% at 28° C with optimum growth at the concentrations of 15 or 20% FBS. Six marine fish viruses (fish nervous necrosis virus, marine birnavirus‐NC1, chum salmon virus, infectious haematopoietic necrosis virus, infectious pancreatic necrosis virus‐Sp and hirame rhabdovirus) were tested on this cell line to determine its susceptibility. After confluency, the cells were subcultured with a split ratio of 1:2. The cells showed epithelial‐like morphology and reached confluency on the fourth day after subculture. Polymerase chain reaction amplification of mitochondrial 12S rRNA indicated identity of these cell lines with those reported from this animal species, confirming that the cell lines were of catla origin. The cells were successfully cryopreserved and revived at passage numbers 10, 25, 40 and 60. The cell cycle analysis by fluorescence‐activated cell sorter revealed that most of the cells on the second day of culture were in S‐phase, indicating a high growth rate. When the SICE cells were transfected with pEGFP vector DNA, significant fluorescent signals were observed suggesting that the SICE cell line can be a useful tool for transgenic and genetic manipulation studies.
A cDNA microarray for the three‐spined stickleback, Gasterosteus aculeatus L., and analysis of the interactive effects of oestradiol and dibenzanthracene exposures
Authors: Geoghegan F. , Katsiadaki I. , Williams T. D. , Chipman J. K.
Published: June 2008

The stickleback is a useful model species for aquatic toxicology and endocrinology and with the recent sequencing of its genome, ecotoxicogenomics. The effects of treatment on male and female sticklebacks with the polycyclic aromatic hydrocarbon (PAH), dibenzanthracene (DbA), the female hormone, 17β‐oestradiol (E2) and a binary mixture of the two were investigated. Quantitative RT‐PCR assays were carried out for cytochrome P450 1A (cyp1A), vitellogenin (VTG) and oestrogen receptor alpha, and cyp1A enzyme activity was assessed by measurement of ethoxyresorufin‐o‐deethylase (EROD) activity , VTG and spiggin protein by enzyme‐linked immunosorbent assay. A stickleback cDNA microarray consisting of 9692 clones was developed and used to assess gene expression responses to the treatments. Induction of cyp1A mRNA and EROD activity was seen in both sexes in response to DbA and a further induction of cyp1A found with the binary treatment. VTG mRNA was induced in male fish exposed to E2 and the binary mixture, while for females, statistically significant induction was seen only with the binary mixture. Similar behaviour was found with zona pellucida and chorion protein mRNA using the microarray. ERα mRNA was induced with E2 and the binary mixture in both sexes. The microarray showed additional transcripts that were differentially regulated by these treatments, which provide novel candidates for biomarker development and mechanistic studies.
A first generation Atlantic halibut Hippoglossus hippoglossus (L.) microarray: application to developmental studies
Authors: Douglas S. E. , Knickle L. C. , Williams J. , Flight R. M. , Reith M. E.
Published: June 2008

An oligonucleotide microarray containing 50‐mer oligonucleotides representing 9277 unique Atlantic halibut Hippoglossus hippoglossus genes has been designed, printed and is currently being used for the study of gene expression in developing halibut. The oligonucleotides are based on all the Atlantic halibut data available at the time of printing, these included expressed sequence tags (ESTs) and complete cDNAs derived from the Pleurogene sequencing project as well as sequences deposited in GenBank by other groups as of September 2006. Of the Pleurogene ESTs, 5040 are functionally annotated; the remainder are unknown (1016) or are similar to unannotated sequences in GenBank (1626). In addition to Atlantic halibut features, several control features have been incorporated, including an oligonucleotide representing a heterologous plant gene (92 spots) and empty spots containing buffer only (1344). The array contains 48 subgrids, each comprised of 32 columns and 26 rows. Every feature is printed at least four times as side‐by‐side quadruplicates, resulting in a microarray containing 39 936 features. This microarray has been utilized to identify genes differentially expressed in larval Atlantic halibut during the developmental period from post‐hatch to post‐metamorphosis. Early in development, transcription of the gene for hatching enzyme was down‐regulated, whereas a gene involved in eye development was up‐regulated. Midway to metamorphosis, transcription of genes encoding several key digestive enzymes was up‐regulated, and in pre‐metamorphic larvae, transcription of genes encoding muscle proteins was prominent.
Identification of development and tissue‐specific gene expression in the fathead minnow Pimephales promelas, Rafinesque using computational and DNA microarray methods
Authors: Kane M. D. , Sringer J. A. , Iannotti N. V. , Gough E. , Johns S. M. , Schlueter S. D. , Sepúlveda M. S.
Published: June 2008

The fathead minnow Pimephales promelas serves as a model organism for assessing the effects of environmental contaminants on early life stage growth and development. Yet, the utilization of genomic tools has been hindered by the lack of genome sequence and genomic information known from this model species. Utilizing published cDNA library sequences, the authors used sequence similarity to compare 4105 cDNAs isolated from fathead minnow fry (<14 days old) with over 250 000 adult cDNA sequences derived from whole body and various tissue types. The objectives of the computational subtraction were to (1) assess the extent of sequence similarity between developing and adult cDNA libraries and (2) predict which cDNA clones are expressed only in developing organisms. The results of the computational predictions were assessed through the construction of a development‐specific DNA microarray targeting all 4105 sequences in the fry cDNA library as well as 56 known mRNAs in P. promelas. Gene expression was determined by comparing total RNA isolated from fry with total RNA isolated from adult samples (whole animal, kidney, liver, brain, ovary and testes). The results showed that 1381 of the targeted fry cDNA sequences (34%) displayed expression across all sample comparisons, and of these, only 166 genes were found to harbour fry‐specific expression (i.e. no expression in adult samples). Of note, 69% of the genes computationally predicted to be fry specific were found across all experimental results; yet, only 27% of the computationally predicted fry‐specific sequences were experimentally confirmed to be fry specific. An important result was the identification of many novel mRNA sequences specific to the developing minnow, which lack homology with any other known sequence. In addition, the study results included tissue‐specific expression in adult samples. These results demonstrate the capabilities and limitations of inter‐library sequence comparisons as a predictor of gene activity in non‐sequenced organisms and tissues, as well as DNA microarray gene expression studies in non‐sequenced organisms.
Altered gene expression in the brain and liver of female fathead minnows Pimephales promelas Rafinesque exposed to fadrozole
Authors: Villeneuve D. L. , Knoebl I. , Larkin P. , Miracle A. L. , Carter B. J. , Denslow N. D. , Ankley G. T.
Published: June 2008

The fathead minnow Pimephales promelas is a small fish species widely used for ecotoxicology research and regulatory testing in North America. This study used a 2000 gene oligonucleotide microarray to evaluate the effects of the aromatase inhibitor, fadrozole, on gene expression in the liver and brain tissue of exposed females. Reproductive measures, plasma vitellogenin and gene expression data for the brain isoform of aromatase (cytP19B), vitellogenin precursors and transferrin provided evidence supporting the efficacy of the fadrozole exposure. Unsupervised analysis of the microarray results identified 20 genes in brain and 41 in liver as significantly up‐regulated and seven genes in brain and around 45 in liver as significantly down‐regulated. Differentially expressed genes were associated with a broad spectrum of biological functions, many with no obvious relationship to aromatase inhibition. However, in brain, fadrozole exposure elicited significant up‐regulation of several genes involved in the cholesterol synthesis, suggesting it as a potentially affected pathway. Gene ontology‐based analysis of expression changes in liver suggested overall down‐regulation of protein biosynthesis. While real‐time polymerase chain reaction analyses supported some of the microarray responses, others could not be verified. Overall, results of this study provide a foundation for developing novel hypotheses regarding the system‐wide effects of fadrozole, and other chemical stressors with similar modes of action, on fish biology.
Utilization of microarray technology for functional genomics in ictalurid catfish
Authors: Liu Z. , Li R. W. , Waldbieser G. C.
Published: June 2008

The channel catfish, Ictalurus punctatus, and the closely related blue catfish, Ictalurus furcatus, are important species in aquaculture and serve as biological models for immunology, neurobiology and environmental monitoring. Directed and high‐throughput sequencing technologies have produced 44 767 channel catfish expressed sequence tags (ESTs) and 10 764 blue catfish ESTs to date. Experiments incorporating large‐scale gene expression analyses have utilized a 660 cDNA macroarray, a 1900 cDNA spotted microarray and in situ synthesized oligonucleotide microarrays covering 19 000 and 28 000 genes. These experiments have revealed numerous transcripts that are differentially expressed in response to environmental stressors, immunostimulation and exposure to viral and bacterial pathogens. A current collaboration with the U.S. Department of Energy’s Community Sequencing Programme will soon provide a much larger resource of ESTs for both catfish species. This will enhance the coverage of the catfish transcriptome and lead to the production of more comprehensive microarrays for future investigations. Functional characterization of the catfish genome will improve the understanding of genetic variation in catfish populations and their responses to environmental variation and enhance the role of the channel catfish as a model species and source of dietary protein.
Use of a 15 k gene microarray to determine gene expression changes in response to acute and chronic methylmercury exposure in the fathead minnow Pimephales promelas Rafinesque
Authors: Klaper R. , Carter B. J. , Richter C. A. , Drevnick P. E. , Sandheinrich M. B. , Tillitt D. E.
Published: June 2008

This study describes the use of a 15 000 gene microarray developed for the toxicological model species, Pimephales promelas, in investigating the impact of acute and chronic methylmercury exposures in male gonad and liver tissues. The results show significant differences in the individual genes that were differentially expressed in response to each treatment. In liver, a total of 650 genes exhibited significantly (P < 0·05) altered expression with greater than two‐fold differences from the controls in response to acute exposure and a total of 267 genes were differentially expressed in response to chronic exposure. A majority of these genes were downregulated rather than upregulated. Fewer genes were altered in gonad than in liver at both timepoints. A total of 212 genes were differentially expressed in response to acute exposure and 155 genes were altered in response to chronic exposure. Despite the differences in individual genes expressed across treatments, the functional categories that altered genes were associated with showed some similarities. Of interest in light of other studies involving the effects of methylmercury on fish, several genes associated with apoptosis were upregulated in response to both acute and chronic exposures. Induction of apoptosis has been associated with effects on reproduction seen in the previous studies. This study demonstrates the utility of microarray analysis for investigations of the physiological effects of toxicants as well as the time‐course of effects that may take place. In addition, it is the first publication to demonstrate the use of this new 15 000 gene microarray for fish biology and toxicology.
The Gillichthys mirabilis Cooper array: a platform to investigate the molecular basis of phenotypic plasticity
Authors: Gracey A. Y.
Published: June 2008

A description of the production of a cDNA microarray platform for the goby Gillichthys mirabilis from an associated collection of expressed sequence tags is presented in this study. The arrayed cDNAs were derived from cDNA libraries prepared from RNA sourced from fish that had been acclimating to a wide variety of environmental conditions. The use of the array is illustrated by a study that investigates the gene expression response of emerged fish that switch from aquatic respiration to air breathing. These results show that the gene expression changes associated with air breathing resemble those observed in fish experiencing aquatic hypoxia suggesting that oxygen requirements are not fully met in animals which have been forced to emerge from sea water to air.
Construction of a robust microarray from a non‐model species largemouth bass, Micropterus salmoides (Lacèpede), using pyrosequencing technology
Authors: Garcia‐Reyero N. , Griffitt R. J. , Liu L. , Kroll K. J. , Farmerie W. G. , Barber D. S. , Denslow N. D.
Published: June 2008

A novel custom microarray for largemouth bass (LMB) Micropterus salmoides was designed with sequences obtained from a normalized cDNA library using the 454 Life Sciences GS‐20 pyrosequencer. This approach yielded in excess of 58 million bases of high‐quality sequence. The sequence information was combined with 2616 reads obtained by traditional suppressive subtractive hybridizations to derive a total of 31 391 unique sequences. Annotation and coding sequences were predicted for these transcripts where possible. About 15 950 annotated transcripts were selected as target sequences for the design of the custom LMB oligonucleotide microarray. The microarray was validated by examining the transcriptomic response in male LMB exposed to 17β‐oestradiol. Transcriptomic responses were assessed in liver and gonad and indicated gene expression profiles typical of exposure to oestradiol. The results demonstrate the potential to rapidly create the tools necessary to assess large‐scale transcriptional responses in non‐model species, paving the way for expanded impact of toxicogenomics in ecotoxicology.
Expansion of the genomics research on Atlantic salmon Salmo salar L. project (GRASP) microarray tools
Authors: VonSchalburg K. R. , Cooper G. A. , Leong J. , Robb A. , Lieph R. , Rise M. L. , Davidson W. S. , Koop B. F.
Published: June 2008

Salmonids are the most widely studied group of fish, and in the last few years, genomics technologies have begun to contribute to this rich biology. The first salmonid microarrays appeared in 2004 and since then several dozen studies have demonstrated the utility of genomic approaches. The widespread use of the genomics research on Atlantic salmon project 16 k array and greatly expanded genome resources have led to the development of an experimental 5 k oligo (70‐mer) array and a 32 k cDNA microarray in the near future. In this paper, the authors examined some of the procedures used in the development of past arrays and reexamined them in light of new genomic data available. Some preliminary control experiments of the new 5 k array were investigated that examine oligo designs based on distance from the polyA tail, the effects of mismatches and cross‐species hybridization specificity. Beneficial approaches are also identified in the development of the new 32 k cDNA array.