Co-ordinate single-cell expression of LEE4- and LEE5-encoded proteins of Escherichia coli O157:H7

Escherichia coli O157:H7 is a zoonotic pathogen that can express a type III secretion system (TTSS) considered important for colonization and persistence in ruminants. E. coli O157:H7 strains have been shown to vary markedly in levels of protein secreted using the TTSS and this study has confirmed that a high secretion phenotype is more prevalent among isolates associated with human disease than isolates shed by healthy cattle. The variation in secretion levels is a consequence of heterogeneous expression, being dependent on the proportion of bacteria in a population that are actively engaged in protein secretion. This was demonstrated by indirect immunofluorescence and eGFP fusions that examined the expression of locus of enterocyte effacement (LEE)-encoded factors in individual bacteria. In liquid media, the expression of EspA, tir::egfp, intimin, but not map::egfp were co-ordinated in a subpopulation of bacteria. In contrast to E. coli O157:H7, expression of tir::egfp in EPEC E2348/69 was equivalent in all bacteria although the same fusion exhibited variable expression when transformed into an E. coli O157:H7 background. An E. coli O157:H7 strain deleted for the LEE demonstrated weak but variable expression of tir::egfp indicating that the elements controlling the heterogeneous expression lie outside the LEE. The research also demonstrated the rapid induction of tir::egfp and map::egfp on contact with bovine epithelial cells. This control in E. coli O157:H7 may be required to limit exposure of key surface antigens, EspA, Tir and intimin during colonization of cattle but allow their rapid production on contact with bovine gastrointestinal epithelium at the terminal rectum.

Evaluation of the contribution of the placentomal fusion during gestation in cattle

The placenta of mammals is a structure formed by the juxtaposition of the fetal membranes and the maternal tissues. The main function of the placenta is to regulate the physiological interchange between the fetus and the mother as well as to operate as an important endocrine organ during the gestation. The placentomal fusions were characterized throughout gestation of cattle using macroscopic, histological and flow cytometry analyses. Analyzing the cell cycle phases with a flow cytometry, a balance between the G2M phase and apoptosis was observed, suggesting that the placentomal fusions do not interfere in the placentary maturation process, which is a pre-requirement for the fetal-maternal disconnection and the release of fetal membrane. (C) 2011 Elsevier B.V. All rights reserved.

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis

Mitotic and meiotic chromosomes of Tityus bahiensis were investigated using light (LM) and transmission electron microscopy (TEM) to determine the chromosomal characteristics and disclose the mechanisms responsible for intraspecific variability in chromosome number and for the presence of complex chromosome association during meiosis. This species is endemic to Brazilian fauna and belongs to the family Buthidae, which is considered phylogenetically basal within the order Scorpiones. In the sample examined, four sympatric and distinct diploid numbers were observed: 2n = 5, 2n = 6, 2n = 9, and 2 = 10. The origin of this remarkable chromosome variability was attributed to chromosome fissions and/or fusions, considering that the decrease in chromosome number was concomitant with the increase in chromosome size and vice versa. The LM and TEM analyses showed the presence of chromosomes without localised centromere, the lack of chiasmata and recombination nodules in male meiosis, and two nucleolar organiser regions carrier chromosomes. Furthermore, male prophase I cells revealed multivalent chromosome associations and/or unsynapsed or distinctly associated chromosome regions (gaps, less-condensed chromatin, or loop-like structure) that were continuous with synapsed chromosome segments. All these data permitted us to suggest that the chromosomal rearrangements of T. bahiensis occurred in a heterozygous state. A combination of various factors, such as correct disjunction and balanced segregation of the chromosomes involved in complex meiotic pairing, system of achiasmate meiosis, holocentric nature of the chromosomes, population structure, and species dispersion patterns, could have contributed to the high level of chromosome rearrangements present in T. bahiensis.

Chromosomal Evolution in the Brazilian Geckos of the Genus Gymnodactylus (Squamata, Phyllodactylidae) from the Biomes of Cerrado, Caatinga and Atlantic Rain Forest: Evidence of Robertsonian Fusion Events and Supernumerary Chromosomes

Chromosomes of the South American geckos Gymnodactylus amarali and G. geckoides from open and dry areas of the Cerrado and Caatinga biomes in Brazil, respectively, were studied for the first time, after conventional and AgNOR staining, CBG- and RBG-banding, and FISH with telomeric sequences. Comparative analyses between the karyotypes of open areas and the previously studied Atlantic forest species G. darwinii were also performed. The chromosomal polymorphisms detected in populations of G. amarali from the states of Goias and Tocantins is the result of centric fusions (2n = 38, 39 and 40), suggesting a differentiation from a 2n = 40 ancestral karyotype and the presence of supernumerary chromosomes. The CBG- and RBG-banding patterns of the Bs are described. G. geckoides has 40 chromosomes with gradually decreasing sizes, but it is distinct from the 2n = 40 karyotypes of G. amarali and G. darwinii due to occurrence of pericentric inversions or centromere repositioning. NOR location seems to be a marker for Gymnodactylus, as G. amarali and G. geckoides share a medium-sized subtelocentric NOR-bearing pair, while G. darwinii has NORs at the secondary constriction of the long arm of pair 1. The comparative analyses indicate a non-random nature of the Robertsonian rearrangements in the genus Gymnodactylus. Copyright (C) 2010 S. Karger AG, Basel

Hematopoietic perturbation in zebrafish expressing a tel-jak2a fusion

Objective
Various TEL-JAK2 fusions have been identified in patients with lymphoblastic and myeloid leukemias that result in constitutive activation of the JAK2 kinase domain. Such fusions can mediate factor-independent growth of hematopoietic cell lines and induction of malignancy in mouse models.
Materials and methods
To assess whether zebrafish could be utilized as a suitable model for the study of myeloid oncogenesis, we generated a zebrafish tel-jak2a fusion oncoprotein based on that seen in a case of chronic myeloid leukemia. This was transiently expressed in zebrafish embryos under the control of the spi1 promoter, which is strongly active in myeloid precursors.
Results
Visual, histological, and molecular analysis revealed disruption of normal embryonic hematopoiesis, including perturbation of the myeloid and erythroid lineages.
Conclusion
These results indicate that the zebrafish tel-jak2a oncoprotein is functional, and suggest that this organism will be useful for the experimental study of myeloid malignancy.

This story wants to be danced : creative writing, dance, convergence and confluence

I suggest ways the discipline of dance can enrich and challenge the discipline of creative writing. I focus particularly on improvisation in dance, relating this to creative writing pedagogy, classroom structure and activities. Much possibility exists in utilising moments when creative arts disciplines touch. How might creative writers and creative writing courses use such fusions? I draw on material theory and briefly upon transformative and collaborative education theories in my exploration of these ideas.

Development of monoclonal antibodies against Vibrio pathogens

Monoclonal antibodies were developed against pathogenic vibrios for use in rapid identification in disease situations of humans, fish and shellfish. Of the 12 fusions performed using V. alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus and V. vulnificus, a total of 102 hybridomas were obtained. Based on cross-reactivity of a wide range of Vibrio strains and other gram-negative bacteria, three broad types of monoclonal antibodies were found. The three categories were: (1) ones that were species-specific or specific to a particular surface antigen, (2) a large number that reacted with several Vibrio species, and (3) three that reacted with most Vibrio strains but no other gram-negative bacteria. Each species-specific monoclonal antibody only recognized its corresponding Vibrio species and was used for identifying unknown species, confirming diagnosis of clinical isolates. In addition, several monoclonal antibodies only cross-reacted with similar Vibrio species, e.g. V. parahaemolyticus and V. alginolyticus which share a common H-antigen. Monoclonal antibodies reacting with several Vibrio species were not of particular use in diagnostic situations. Three monoclonal antibodies of the last group did not react with other genera of the family Vibrionaceae, namely Aeromonas, Photobacterium and Plesiomonas nor a wide range of gram-negative enteric bacteria. These data indicated the existence of an antigenic surface determinant common to Vibrio species. One monoclonal reacted with the heat-stable antigenic determinants on the cell surface as v as lipopolysaccharide extracted from all the vibrios studied, thus making it useful for large- scale screening of acute infections of vibrios. In a blind test, seven Vibrio species, isolated from 6 marine and a freshwater source were identified by two laboratories using phenetic tests. Results of immunotyping using monoclonals, three of seven were diagnosed as the same species, another three were designated as Vibrio species but could not be classified further due to the library not having the corresponding monoclonal, and one was diagnostically questionable. Two further tests were carried out. An unknown Vibrio formalin-fixed isolated from diseased marine animal was identified as V. parahaemolyticus by ELISA and FITC. Clinical human isolates of V. alginolyticus, V. parahaemolyticus and V. vulnificus were confirmed by monoclonals. Australian isolates of V. anguillarum appeared to be mostly of serotype O1. monoclonals raised to V. anguillarum AFHRL 1 reacted with only serotype O1 from Denmark but also most Australian isolates. All vibrios pathogenic to fish and shellfish, i.e. V. anguillarum, V. ordalii, V. alginolyticus, V. carchariae, V. cholerae, V. damsela, V. harveyi, V. parahaemolyticus and V. vulnificus, were used for attachment studies to fish cells using phase contrast and FITC-immunofluorescence microscopy. Of these vibrios, V. anguillarum, V. ordalii and V. perahaemolyticus, were found to adhere to different cells and tissues of rainbow trout while others did not appear to attach. However, attachment was inhibited by monoclonal antibodies specific to only these three vibrios. Lipopolysaccharide is well known as being a contributing factor in pathogenicity of gram-negative bacteria. PAGE electrophoresis of extracted LPS from 9 strains covering 6 Vibrio species showed the presence of a common 15,000 D fragment. This fragment was verified by immunoblotting with a genus-specific monoclonal antibody (i.e. F11P411F) recognizing nearly all vibrios. The common LPS fragment was separated and used to raise polyclonal antisera in mouse which reacted strongly with LPS itself, live as well as sodium azide-killed vibrios, but not with other gram-negative bacteria. This raised the possibility of developing vaccine from Vibrio LPS. Monoclonal antibodies developed in the present study enabled rapid identification of a number of pathogenic Vibrio species. There is still further work to produce monoclonal antibodies against additional vibrios that are probably pathogenic. These included V. fluvialis, V. hollisae, V. metschnikovii, V. minicus, V. salmonella and V. tubiashii. Together the application will be of significance in clinical diagnostic work, in the monitoring of vibriosis in fish farms and in quarantine.

Modeling intelligent agents for web-based information gathering

The recent emergence of intelligent agent technology and advances in information gathering have been the important steps forward in efficiently managing and using the vast amount of information now available on the Web to make informed decisions. There are, however, still many problems that need to be overcome in the information gathering research arena to enable the delivery of relevant information required by end users. Good decisions cannot be made without sufficient, timely, and correct information. Traditionally it is said that knowledge is power, however, nowadays sufficient, timely, and correct information is power. So gathering relevant information to meet user information needs is the crucial step for making good decisions. The ideal goal of information gathering is to obtain only the information that users need (no more and no less). However, the volume of information available, diversity formats of information, uncertainties of information, and distributed locations of information (e.g. World Wide Web) hinder the process of gathering the right information to meet the user needs. Specifically, two fundamental issues in regard to efficiency of information gathering are mismatch and overload. The mismatch means some information that meets user needs has not been gathered (or missed out), whereas, the overload means some gathered information is not what users need. Traditional information retrieval has been developed well in the past twenty years. The introduction of the Web has changed people's perceptions of information retrieval. Usually, the task of information retrieval is considered to have the function of leading the user to those documents that are relevant to his/her information needs. The similar function in information retrieval is to filter out the irrelevant documents (or called information filtering). Research into traditional information retrieval has provided many retrieval models and techniques to represent documents and queries. Nowadays, information is becoming highly distributed, and increasingly difficult to gather. On the other hand, people have found a lot of uncertainties that are contained in the user information needs. These motivate the need for research in agent-based information gathering. Agent-based information systems arise at this moment. In these kinds of systems, intelligent agents will get commitments from their users and act on the users behalf to gather the required information. They can easily retrieve the relevant information from highly distributed uncertain environments because of their merits of intelligent, autonomy and distribution. The current research for agent-based information gathering systems is divided into single agent gathering systems, and multi-agent gathering systems. In both research areas, there are still open problems to be solved so that agent-based information gathering systems can retrieve the uncertain information more effectively from the highly distributed environments. The aim of this thesis is to research the theoretical framework for intelligent agents to gather information from the Web. This research integrates the areas of information retrieval and intelligent agents. The specific research areas in this thesis are the development of an information filtering model for single agent systems, and the development of a dynamic belief model for information fusion for multi-agent systems. The research results are also supported by the construction of real information gathering agents (e.g., Job Agent) for the Internet to help users to gather useful information stored in Web sites. In such a framework, information gathering agents have abilities to describe (or learn) the user information needs, and act like users to retrieve, filter, and/or fuse the information. A rough set based information filtering model is developed to address the problem of overload. The new approach allows users to describe their information needs on user concept spaces rather than on document spaces, and it views a user information need as a rough set over the document space. The rough set decision theory is used to classify new documents into three regions: positive region, boundary region, and negative region. Two experiments are presented to verify this model, and it shows that the rough set based model provides an efficient approach to the overload problem. In this research, a dynamic belief model for information fusion in multi-agent environments is also developed. This model has a polynomial time complexity, and it has been proven that the fusion results are belief (mass) functions. By using this model, a collection fusion algorithm for information gathering agents is presented. The difficult problem for this research is the case where collections may be used by more than one agent. This algorithm, however, uses the technique of cooperation between agents, and provides a solution for this difficult problem in distributed information retrieval systems. This thesis presents the solutions to the theoretical problems in agent-based information gathering systems, including information filtering models, agent belief modeling, and collection fusions. It also presents solutions to some of the technical problems in agent-based information systems, such as document classification, the architecture for agent-based information gathering systems, and the decision in multiple agent environments. Such kinds of information gathering agents will gather relevant information from highly distributed uncertain environments.

A lightweight intrusion alert fusion system

In this paper, we present some practical experience on implementing an alert fusion mechanism from our project. After investigation on most of the existing alert fusion systems, we found the current body of work alternatively weighed down in the mire of insecure design or rarely deployed because of their complexity. As confirmed by our experimental analysis, unsuitable mechanisms could easily be submerged by an abundance of useless alerts. Even with the use of methods that achieve a high fusion rate and low false positives, attack is also possible. To find the solution, we carried out analysis on a series of alerts generated by well-known datasets as well as realistic alerts from the Australian Honey-Pot. One important finding is that one alert has more than an 85% chance of being fused in the following 5 alerts. Of particular importance is our design of a novel lightweight Cache-based Alert Fusion Scheme, called CAFS. CAFS has the capacity to not only reduce the quantity of useless alerts generated by IDS (Intrusion Detection System), but also enhance the accuracy of alerts, therefore greatly reducing the cost of fusion processing. We also present reasonable and practical specifications for the target-oriented fusion policy that provides a quality guarantee on alert fusion, and as a result seamlessly satisfies the process of successive correlation. Our experimental results showed that the CAFS easily attained the desired level of survivable, inescapable alert fusion design. Furthermore, as a lightweight scheme, CAFS can easily be deployed and excel in a large amount of alert fusions, which go towards improving the usability of system resources. To the best of our knowledge, our work is a novel exploration in addressing these problems from a survivable, inescapable and deployable point of view.

CAFS : a novel lightweight cache-based scheme for large-scale intrusion alert fusion

In this paper, we present some practical experiences on implementing an alert fusion mechanism from our project. After investigation on most of the existing alert fusion systems, we found the current body of work alternatively weighed down in the mire of insecure design or rarely deployed because of their complexity. As confirmed by our experimental analysis, unsuitable mechanisms could easily be submerged by an abundance of useless alerts. Even with the use of methods that achieve a high fusion rate and low false positives, attack is also possible. To find the solution, we carried out analysis on a series of alerts generated by well-known datasets as well as realistic alerts from the Australian Honey-Pot. One important finding is that one alert has more than an 85% chance of being fused in the following five alerts. Of particular importance is our design of a novel lightweight Cache-based Alert Fusion Scheme, called CAFS. CAFS has the capacity to not only reduce the quantity of useless alerts generated by intrusion detection system, but also enhance the accuracy of alerts, therefore greatly reducing the cost of fusion processing. We also present reasonable and practical specifications for the target-oriented fusion policy that provides a quality guarantee on alert fusion, and as a result seamlessly satisfies the process of successive correlation. Our experiments compared CAFS with traditional centralized fusion. The results showed that the CAFS easily attained the desired level of simple, counter-escapable alert fusion design. Furthermore, as a lightweight scheme, CAFS can easily be deployed and excel in a large amount of alert fusions, which go towards improving the usability of system resources. To the best of our knowledge, our work is a practical exploration in addressing problems from the academic point of view. Copyright © 2011 John Wiley & Sons, Ltd.

D. A. Pennebaker

This volume is the first book-length study of the extensive career and prolific works of D.A. Pennebaker, one of the pioneers of direct cinema, a documentary form that emphasizes observation and a straightforward portrayal of events. With a career spanning decades, Pennebaker's many projects have included avant-garde experiments (Daybreak Express), ground-breaking television documentaries (Primary), celebrity films (Dont Look Back), concert films (Monterey Pop), and innovative fusions of documentary and fiction (Maidstone).

Exploring the concept of "performing the real," Keith Beattie's insightful analysis interprets Pennebaker's films as performances in which the act of filming is in itself a performative transgression of the norms of purely observational documentary. He examines the ways in which Pennebaker's presentation of unscripted everyday performances is informed by connections between documentary filmmaking and other experimental movements such as the New American Cinema. Through his collaborations with such various artists as Richard Leacock, Shirley Clarke, Norman Mailer, and Jean-Luc Godard, Pennebaker has continually reworked and redefined the forms of documentary filmmaking. This book also includes a recent interview with the director and a full filmography.

"A welcome addition to the Contemporary Film Directors Series."--Documentary

"Filled with useful historical and technical details, this enthusiastic study of one of documentary cinema's most important filmmakers will convert some skeptics and create many new admirers of D.A. Pennebaker's work."--Joe McElhaney, author of Albert Maysles.

Investigation of the plasmodium falciparum food vacuole through inducible expression of the chloroquine resistance transporter (PfCRT)

Haemoglobin degradation during the erythrocytic life stages is the major function of the food vacuole (FV) of Plasmodium falciparum and the target of several anti-malarial drugs that interfere with this metabolic pathway, killing the parasite. Two multi-spanning food vacuole membrane proteins are known, the multidrug resistance protein 1 (PfMDR1) and Chloroquine Resistance Transporter (PfCRT). Both modulate resistance to drugs that act in the food vacuole. To investigate the formation and behaviour of the food vacuole membrane we have generated inducible GFP fusions of chloroquine sensitive and resistant forms of the PfCRT protein. The inducible expression system allowed us to follow newly-induced fusion proteins, and corroborated a previous report of a direct trafficking route from the ER/Golgi to the food vacuole membrane. These parasites also allowed the definition of a food vacuole compartment in ring stage parasites well before haemozoin crystals were apparent, as well as the elucidation of secondary PfCRT-labelled compartments adjacent to the food vacuole in late stage parasites. We demonstrated that in addition to previously demonstrated Brefeldin A sensitivity, the trafficking of PfCRT is disrupted by Dynasore, a non competitive inhibitor of dynamin-mediated vesicle formation. Chloroquine sensitivity was not altered in parasites over-expressing chloroquine resistant or sensitive forms of the PfCRT fused to GFP, suggesting that the PfCRT does not mediate chloroquine transport as a GFP fusion protein.

ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish

Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal function of ETV6 remains incomplete. This study has employed zebrafish as a relevant model to investigate the role of ETV6 during embryonic hematopoiesis. Zebrafish possessed a single conserved etv6 ortholog that was expressed from 12 hpf in the lateral plate mesoderm, and later in hematopoietic, vascular and other tissues. Morpholino-mediated gene knockdown of etv6 revealed the complex contribution of this gene toward embryonic hematopoiesis. During primitive hematopoiesis, etv6 knockdown resulted in reduced levels of progenitor cells, erythrocyte and macrophage populations, but increased numbers of incompletely differentiated heterophils. Definitive hematopoiesis was also perturbed, with etv6 knockdown leading to decreased erythrocytes and myeloid cells, but enhanced lymphopoiesis. This study suggests that ETV6 plays a broader and more complex role in early hematopoiesis than previously thought, impacting on the development of multiple lineages. © 2015 Ferrata Storti Foundation.

Dissecting the role of the ETV6 transcriptional regulator in development and disease

This study studied the ETV6 gene using zebrafish as a model. The work demonstrated that ETV6 was important for the development of both white and red blood cells. It also characterized the pathogenic effects of mutant forms of ETV6 found in various leukemias and other blood disorders.

Alzheimer's Abeta fused to green fluorescent protein induces growth stress and a heat shock response

The 42 amino acid Alzheimer's Aβ peptide is involved in the progression of Alzheimer's disease. Here we describe the effects of intracellular Aβ, produced through its attachment to either end of a green fluorescent protein, in yeast. Cells producing Aβ exhibited a lower growth yield and a heat shock response, showing that Aβ fusions promote stress in cells and supporting the notion that intracellular Aβ is a toxic molecule. These studies have relevance in understanding the role of Aβ in the death of neuronal cells, and indicate that yeast may be a new tractable model system for the screening for inhibitors of the stress caused by Aβ.