Optimisation of Bioluminescent Reporters for Use with Mycobacteria

BACKGROUND: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI. METHODOLOGY/PRINCIPAL FINDINGS: One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging. CONCLUSIONS/SIGNIFICANCE: While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase

Female Burying Beetles Benefit from Male Desertion: Sexual Conflict and Counter-Adaptation over Parental Investment

Sexual conflict drives the coevolution of sexually antagonistic traits, such that an adaptation in one sex selects an opposing coevolutionary response from the other. Although many adaptations and counteradaptations have been identified in sexual conflict over mating interactions, few are known for sexual conflict over parental investment. Here we investigate a possible coevolutionary sequence triggered by mate desertion in the burying beetle Nicrophorus vespilloides, where males commonly leave before their offspring reach independence. Rather than suffer fitness costs as a consequence, our data suggest that females rely on the male's absence to recoup some of the costs of larval care, presumably because they are then free to feed themselves on the carcass employed for breeding. Consequently, forcing males to stay until the larvae disperse reduces components of female fitness to a greater extent than caring for young singlehandedly. Therefore we suggest that females may have co-evolved to anticipate desertion by their partners so that they now benefit from the male's absence

Continuous Interaction with a Virtual Human

Attentive Speaking and Active Listening require that a Virtual Human be capable of simultaneous perception/interpretation and production of communicative behavior. A Virtual Human should be able to signal its attitude and attention while it is listening to its interaction partner, and be able to attend to its interaction partner while it is speaking – and modify its communicative behavior on-the-fly based on what it perceives from its partner. This report presents the results of a four week summer project that was part of eNTERFACE’10. The project resulted in progress on several aspects of continuous interaction such as scheduling and interrupting multimodal behavior, automatic classification of listener responses, generation of response eliciting behavior, and models for appropriate reactions to listener responses. A pilot user study was conducted with ten participants. In addition, the project yielded a number of deliverables that are released for public access

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease

Galaxy evolution: black hole feedback in the luminous quasar PDS 456

The evolution of galaxies is connected to the growth of supermassive black holes in their centers. During the quasar phase, a huge luminosity is released as matter falls onto the black hole, and radiation-driven winds can transfer most of this energy back to the host galaxy. Over five different epochs, we detected the signatures of a nearly spherical stream of highly ionized gas in the broadband x-ray spectra of the luminous quasar PDS 456. This persistent wind is expelled at relativistic speeds from the inner accretion disk, and its wide aperture suggests an effective coupling with the ambient gas. The outflow's kinetic power larger than 10(46) ergs per second is enough to provide the feedback required by models of black hole and host galaxy coevolution

An Interprofessional Education Workshop Integrating Pharmacy, Physical Therapy, Occupational Therapy, and Physician Assistant Students and Faculty

Jefferson Seminar Purpose •This seminar will describe a case-based interprofessional education (IPE) workshop used to facilitate interprofessional learning –Planning –Implementation –Assessmen

Phylogeography of Japanese encephalitis virus:genotype is associated with climate

The circulation of vector-borne zoonotic viruses is largely determined by the overlap in the geographical distributions of virus-competent vectors and reservoir hosts. What is less clear are the factors influencing the distribution of virus-specific lineages. Japanese encephalitis virus (JEV) is the most important etiologic agent of epidemic encephalitis worldwide, and is primarily maintained between vertebrate reservoir hosts (avian and swine) and culicine mosquitoes. There are five genotypes of JEV: GI-V. In recent years, GI has displaced GIII as the dominant JEV genotype and GV has re-emerged after almost 60 years of undetected virus circulation. JEV is found throughout most of Asia, extending from maritime Siberia in the north to Australia in the south, and as far as Pakistan to the west and Saipan to the east. Transmission of JEV in temperate zones is epidemic with the majority of cases occurring in summer months, while transmission in tropical zones is endemic and occurs year-round at lower rates. To test the hypothesis that viruses circulating in these two geographical zones are genetically distinct, we applied Bayesian phylogeographic, categorical data analysis and phylogeny-trait association test techniques to the largest JEV dataset compiled to date, representing the envelope (E) gene of 487 isolates collected from 12 countries over 75 years. We demonstrated that GIII and the recently emerged GI-b are temperate genotypes likely maintained year-round in northern latitudes, while GI-a and GII are tropical genotypes likely maintained primarily through mosquito-avian and mosquito-swine transmission cycles. This study represents a new paradigm directly linking viral molecular evolution and climate

Socioeconomic disparities in breast cancer survival: relation to stage at diagnosis, treatment and race

<p>Abstract</p> <p>Background</p> <p>Previous studies have documented lower breast cancer survival among women with lower socioeconomic status (SES) in the United States. In this study, I examined the extent to which socioeconomic disparity in breast cancer survival was explained by stage at diagnosis, treatment, race and rural/urban residence using the Surveillance, Epidemiology, and End Results (SEER) data.</p> <p>Methods</p> <p>Women diagnosed with breast cancer during 1998-2002 in the 13 SEER cancer registry areas were followed-up to the end of 2005. The association between an area-based measure of SES and cause-specific five-year survival was estimated using Cox regression models. Six models were used to assess the extent to which SES differences in survival were explained by clinical and demographical factors. The base model estimated the hazard ratio (HR) by SES only and then additional adjustments were made sequentially for: 1) age and year of diagnosis; 2) stage at diagnosis; 3) first course treatment; 4) race; and 5) rural/urban residence.</p> <p>Results</p> <p>An inverse association was found between SES and risk of dying from breast cancer (p < 0.0001). As area-level SES falls, HR rises (1.00 → 1.05 → 1.23 → 1.31) with the two lowest SES groups having statistically higher HRs. This SES differential completely disappeared after full adjustment for clinical and demographical factors (p = 0.20).</p> <p>Conclusion</p> <p>Stage at diagnosis, first course treatment and race explained most of the socioeconomic disparity in breast cancer survival. Targeted interventions to increase breast cancer screening and treatment coverage in patients with lower SES could reduce much of socioeconomic disparity.</p

Potential conservation of circadian clock proteins in the phylum Nematoda as revealed by bioinformatic searches

Although several circadian rhythms have been described in C. elegans, its molecular clock remains elusive. In this work we employed a novel bioinformatic approach, applying probabilistic methodologies, to search for circadian clock proteins of several of the best studied circadian model organisms of different taxa (Mus musculus, Drosophila melanogaster, Neurospora crassa, Arabidopsis thaliana and Synechoccocus elongatus) in the proteomes of C. elegans and other members of the phylum Nematoda. With this approach we found that the Nematoda contain proteins most related to the core and accessory proteins of the insect and mammalian clocks, which provide new insights into the nematode clock and the evolution of the circadian system.Fil: Romanowski, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; ArgentinaFil: Garavaglia, Matías Javier. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Goya, María Eugenia. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ing.genética y Biolog.molecular y Celular. Area Virus de Insectos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Golombek, Diego Andres. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Cronobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Dissecting complex transcriptional responses using pathway-level scores based on prior information

<p>Abstract</p> <p>Background</p> <p>The genomewide pattern of changes in mRNA expression measured using DNA microarrays is typically a complex superposition of the response of multiple regulatory pathways to changes in the environment of the cells. The use of prior information, either about the function of the protein encoded by each gene, or about the physical interactions between regulatory factors and the sequences controlling its expression, has emerged as a powerful approach for dissecting complex transcriptional responses.</p> <p>Results</p> <p>We review two different approaches for combining the noisy expression levels of multiple individual genes into robust pathway-level differential expression scores. The first is based on a comparison between the distribution of expression levels of genes within a predefined gene set and those of all other genes in the genome. The second starts from an estimate of the strength of genomewide regulatory network connectivities based on sequence information or direct measurements of protein-DNA interactions, and uses regression analysis to estimate the activity of gene regulatory pathways. The statistical methods used are explained in detail.</p> <p>Conclusion</p> <p>By avoiding the thresholding of individual genes, pathway-level analysis of differential expression based on prior information can be considerably more sensitive to subtle changes in gene expression than gene-level analysis. The methods are technically straightforward and yield results that are easily interpretable, both biologically and statistically.</p