RNASeqBrowser: A genome browser for simultaneous visualization of raw strand specific RNAseq reads and UCSC genome browser custom tracks

Background Strand specific RNAseq data is now more common in RNAseq projects. Visualizing RNAseq data has become an important matter in Analysis of sequencing data. The most widely used visualization tool is the UCSC genome browser that introduced the custom track concept that enabled researchers to simultaneously visualize gene expression at a particular locus from multiple experiments. Our objective of the software tool is to provide friendly interface for visualization of RNAseq datasets. Results This paper introduces a visualization tool (RNASeqBrowser) that incorporates and extends the functionality of the UCSC genome browser. For example, RNASeqBrowser simultaneously displays read coverage, SNPs, InDels and raw read tracks with other BED and wiggle tracks -- all being dynamically built from the BAM file. Paired reads are also connected in the browser to enable easier identification of novel exon/intron borders and chimaeric transcripts. Strand specific RNAseq data is also supported by RNASeqBrowser that displays reads above (positive strand transcript) or below (negative strand transcripts) a central line. Finally, RNASeqBrowser was designed for ease of use for users with few bioinformatic skills, and incorporates the features of many genome browsers into one platform. Conclusions The features of RNASeqBrowser: (1) RNASeqBrowser integrates UCSC genome browser and NGS visualization tools such as IGV. It extends the functionality of the UCSC genome browser by adding several new types of tracks to show NGS data such as individual raw reads, SNPs and InDels. (2) RNASeqBrowser can dynamically generate RNA secondary structure. It is useful for identifying non-coding RNA such as miRNA. (3) Overlaying NGS wiggle data is helpful in displaying differential expression and is simple to implement in RNASeqBrowser. (4) NGS data accumulates a lot of raw reads. Thus, RNASeqBrowser collapses exact duplicate reads to reduce visualization space. Normal PC’s can show many windows of NGS individual raw reads without much delay. (5) Multiple popup windows of individual raw reads provide users with more viewing space. This avoids existing approaches (such as IGV) which squeeze all raw reads into one window. This will be helpful for visualizing multiple datasets simultaneously. RNASeqBrowser and its manual are freely available at http://www.australianprostatecentre.org/research/software/rnaseqbrowser webcite or http://sourceforge.net/projects/rnaseqbrowser/ webcite

Overcoming language barriers in healthcare: A protocol for investigating safe and effective communication when patients or clinicians use a second language

Background Miscommunication in the healthcare sector can be life-threatening. The rising number of migrant patients and foreign-trained staff means that communication errors between a healthcare practitioner and patient when one or both are speaking a second language are increasingly likely. However, there is limited research that addresses this issue systematically. This protocol outlines a hospital-based study examining interactions between healthcare practitioners and their patients who either share or do not share a first language. Of particular interest are the nature and efficacy of communication in language-discordant conversations, and the degree to which risk is communicated. Our aim is to understand language barriers and miscommunication that may occur in healthcare settings between patients and healthcare practitioners, especially where at least one of the speakers is using a second (weaker) language. Methods/Design Eighty individual interactions between patients and practitioners who speak either English or Chinese (Mandarin or Cantonese) as their first language will be video recorded in a range of in- and out-patient departments at three hospitals in the Metro South area of Brisbane, Australia. All participants will complete a language background questionnaire. Patients will also complete a short survey rating the effectiveness of the interaction. Recordings will be transcribed and submitted to both quantitative and qualitative analyses to determine elements of the language used that might be particularly problematic and the extent to which language concordance and discordance impacts on the quality of the patient-practitioner consultation. Discussion Understanding the role that language plays in creating barriers to healthcare is critical for healthcare systems that are experiencing an increasing range of culturally and linguistically diverse populations both amongst patients and practitioners. The data resulting from this study will inform policy and practical solutions for communication training, provide an agenda for future research, and extend theory in health communication.

A model to explain specific cellular communications and cellular harmony: - a hypothesis of coupled cells and interactive coupling molecules

Background The various cell types and their relative numbers in multicellular organisms are controlled by growth factors and related extracellular molecules which affect genetic expression pathways. However, these substances may have both/either inhibitory and/or stimulatory effects on cell division and cell differentiation depending on the cellular environment. It is not known how cells respond to these substances in such an ambiguous way. Many cellular effects have been investigated and reported using cell culture from cancer cell lines in an effort to define normal cellular behaviour using these abnormal cells. A model is offered to explain the harmony of cellular life in multicellular organisms involving interacting extracellular substances. Methods A basic model was proposed based on asymmetric cell division and evidence to support the hypothetical model was accumulated from the literature. In particular, relevant evidence was selected for the Insulin-Like Growth Factor system from the published data, especially from certain cell lines, to support the model. The evidence has been selective in an attempt to provide a picture of normal cellular responses, derived from the cell lines. Results The formation of a pair of coupled cells by asymmetric cell division is an integral part of the model as is the interaction of couplet molecules derived from these cells. Each couplet cell will have a receptor to measure the amount of the couplet molecule produced by the other cell; each cell will be receptor-positive or receptor-negative for the respective receptors. The couplet molecules will form a binary complex whose level is also measured by the cell. The hypothesis is heavily supported by selective collection of circumstantial evidence and by some direct evidence. The basic model can be expanded to other cellular interactions. Conclusions These couplet cells and interacting couplet molecules can be viewed as a mechanism that provides a controlled and balanced division-of-labour between the two progeny cells, and, in turn, their progeny. The presence or absence of a particular receptor for a couplet molecule will define a cell type and the presence or absence of many such receptors will define the cell types of the progeny within cell lineages.

Comparative kinomics of human and chimpanzee reveal unique kinship and functional diversity generated by new domain combinations

Background: Phosphorylation by protein kinases is a common event in many cellular processes. Further, many kinases perform specialized roles and are regulated by non-kinase domains tethered to kinase domain. Perturbation in the regulation of kinases leads to malignancy. We have identified and analysed putative protein kinases encoded in the genome of chimpanzee which is a close evolutionary relative of human. Result: The shared core biology between chimpanzee and human is characterized by many orthologous protein kinases which are involved in conserved pathways. Domain architectures specific to chimp/human kinases have been observed. Chimp kinases with unique domain architectures are characterized by deletion of one or more non-kinase domains in the human kinases. Interestingly, counterparts of some of the multi-domain human kinases in chimp are characterized by identical domain architectures but with kinase-like non-kinase domain. Remarkably, out of 587 chimpanzee kinases no human orthologue with greater than 95% sequence identity could be identified for 160 kinases. Variations in chimpanzee kinases compared to human kinases are brought about also by differences in functions of domains tethered to the catalytic kinase domain. For example, the heterodimer forming PB1 domain related to the fold of ubiquitin/Ras-binding domain is seen uniquely tethered to PKC-like chimpanzee kinase. Conclusion: Though the chimpanzee and human are evolutionary very close, there are chimpanzee kinases with no close counterpart in the human suggesting differences in their functions. This analysis provides a direction for experimental analysis of human and chimpanzee protein kinases in order to enhance our understanding on their specific biological roles.

Japanese Video Game Localization : A Case Study of Sony's Sairen Series

This thesis is a comparative case study in Japanese video game localization for the video games Sairen, Sairen 2 and Sairen Nyûtoransurêshon, and English-language localized versions of the same games as published in Scandinavia and Australia/New Zealand. All games are developed by Sony Computer Entertainment Inc. and published exclusively for Playstation2 and Playstation3 consoles. The fictional world of the Sairen games draws much influence from Japanese history, as well as from popular and contemporary culture, and in doing so caters mainly to a Japanese audience. For localization, i.e. the adaptation of a product to make it accessible to users outside the original market it was intended for in the first place, this is a challenging issue. Video games are media of entertainment, and therefore localization practice must preserve the games’ effects on the players’ emotions. Further, video games are digital products that are comprised of a multitude of distinct elements, some of which are part of the game world, while others regulate the connection between the player as part of the real world and the game as digital medium. As a result, video game localization is also a practice that has to cope with the technical restrictions that are inherent to the medium. The main theory used throughout the thesis is Anthony Pym’s framework for localization studies that considers the user of the localized product as a defining part of the localization process. This concept presupposes that localization is an adaptation that is performed to make a product better suited for use during a specific reception situation. Pym also addresses the factor that certain products may resist distribution into certain reception situations because of their content, and that certain aspects of localization aim to reduce this resistance through significant alterations of the original product. While Pym developed his ideas with mainly regular software in mind, they can also be adapted well to study video games from a localization angle. Since modern video games are highly complex entities that often switch between interactive and non-interactive modes, Pym’s ideas are adapted throughout the thesis to suit the particular elements being studied. Instances analyzed in this thesis include menu screens, video clips, in-game action and websites. The main research questions focus on how the games’ rules influence localization, and how the games’ fictional domain influences localization. Because there are so many peculiarities inherent to the medium of the video game, other theories are introduced as well to complement the research at hand. These include Lawrence Venuti’s discussions of foreiginizing and domesticating translation methods for literary translation, and Jesper Juul’s definition of games. Additionally, knowledge gathered from interviews with video game localization professionals in Japan during September and October 2009 is also utilized for this study. Apart from answering the aforementioned research questions, one of this thesis’ aims is to enrich the still rather small field of game localization studies, and the study of Japanese video games in particular, one of Japan’s most successful cultural exports.

Access path query language for relational database systems

Database management systems offer a very reliable and attractive data organization for fast and economical information storage and processing for diverse applications. It is much more important that the information should be easily accessible to users with varied backgrounds, professional as well as casual, through a suitable data sublanguage. The language adopted here (APPLE) is one such language for relational database systems and is completely nonprocedural and well suited to users with minimum or no programming background. This is supported by an access path model which permits the user to formulate completely nonprocedural queries expressed solely in terms of attribute names. The data description language (DDL) and data manipulation language (DML) features of APPLE are also discussed. The underlying relational database has been implemented with the help of the DATATRIEVE-11 utility for record and domain definition which is available on the PDP-11/35. The package is coded in Pascal and MACRO-11. Further, most of the limitations of the DATATRIEVE-11 utility have been eliminated in the interface package.

The Evolution of Guanylyl Cyclases as Multidomain Proteins: Conserved Features of Kinase-Cyclase Domain Fusions

Guanylyl cyclases (GCs) are enzymes that generate cyclic GMP and regulate different physiologic and developmental processes in a number of organisms. GCs possess sequence similarity to class III adenylyl cyclases (ACs) and are present as either membrane-bound receptor GCs or cytosolic soluble GCs. We sought to determine the evolution of GCs using a large-scale bioinformatic analysis and found multiple lineage-specific expansions of GC genes in the genomes of many eukaryotes. Moreover, a few GC-like proteins were identified in prokaryotes, which come fused to a number of different domains, suggesting allosteric regulation of nucleotide cyclase activity Eukaryotic receptor GCs are associated with a kinase homology domain (KHD), and phylogenetic analysis of these proteins suggest coevolution of the KHD and the associated cyclase domain as well as a conservation of the sequence and the size of the linker region between the KHD and the associated cyclase domain. Finally, we also report the existence of mimiviral proteins that contain putative active kinase domains associated with a cyclase domain, which could suggest early evolution of the fusion of these two important domains involved in signa transduction.

L2-stability of nonstationary feedback systems: Frequency-domain criteria

A frequency-domain positivity condition is derived for linear time-varying operators in2and is used to develop2stability criteria for linear and nonlinear feedback systems. These criteria permit the use of a very general class of operators in2with nonstationary kernels, as multipliers. More specific results are obtained by using a first-order differential operator with a time-varying coefficient as multiplier. Finally, by employing periodic multipliers, improved stability criteria are derived for the nonlinear damped Mathieu equation with a forcing function.

Finding My Way: Protocol of a randomised controlled trial evaluating an internet self-help program for cancer-related distress

Background A cancer diagnosis elicits greater distress than any other medical diagnosis, and yet very few studies have evaluated the efficacy of structured online self-help therapeutic programs to alleviate this distress. This study aims to assess the efficacy over time of an internet Cognitive Behaviour Therapy (iCBT) intervention (‘Finding My Way’) in improving distress, coping and quality of life for individuals with a recent diagnosis of early stage cancer of any type. Methods/Design The study is a multi-site Randomised Controlled Trial (RCT) seeking to enrol 188 participants who will be randomised to either the Finding My Way Intervention or an attention-control condition. Both conditions are delivered online; with 6 modules released once per week, and an additional booster module released one month after program-completion. Participants complete online questionnaires on 4 occasions: at baseline (immediately prior to accessing the modules); post-treatment (immediately after program-completion); then three and six months later. Primary outcomes are general distress and cancer-specific distress, with secondary outcomes including Health-Related Quality of Life (HRQoL), coping, health service utilisation, intervention adherence, and user satisfaction. A range of baseline measures will be assessed as potential moderators of outcomes. Eligible participants are individuals recently diagnosed with any type of cancer, being treated with curative intent, aged over 18 years with sufficient English language literacy, internet access and an active email account and phone number. Participants are blinded to treatment group allocation. Randomisation is computer generated and stratified by gender. Discussion Compared to the few prior published studies, Finding My Way will be the first adequately powered trial to offer an iCBT intervention to curatively treated patients of heterogeneous cancer types in the immediate post-diagnosis/treatment period. If found efficacious, Finding My Way will assist with overcoming common barriers to face-to-face therapy in a cost-effective and accessible way, thus helping to reduce distress after cancer diagnosis and consequently decrease the cancer burden for individuals and the health system. Trial registration Australian New Zealand Clinical Trials Registry ACTRN12613000001​796 16.10.13

Transitioning from university to the workplace: Stakeholder perceptions of academic and professional writing demands

This study provides a detailed insight into the changing writing demands from the last year of university study to the first year in the workforce of engineering and accounting professionals. The study relates these to the demands of the writing component of IELTS, which is increasingly used for exit testing. The number of international and local students whose first language is not English and who are studying in English-medium universities has increased significantly in the past decade. Many of these students aim to start working in the country they studied in; however, some employers have suggested that graduates seeking employment have insufficient language skills. This study provides a detailed insight into the changing writing demands from the last year of university study to the first year in the workforce of engineering and accounting professionals (our two case study professions). It relates these to the demands of the writing component of IELTS, which is increasingly used for exit or professional entry testing, although not expressly designed for this purpose. Data include interviews with final year students, lecturers, employers and new graduates in their first few years in the workforce, as well as professional board members. Employers also reviewed both final year assignments, as well as IELTS writing samples at different levels. Most stakeholders agreed that graduates entering the workforce are underprepared for the writing demands in their professions. When compared with the university writing tasks, the workplace writing expected of new graduates was perceived as different in terms of genre, the tailoring of a text for a specific audience, and processes of review and editing involved. Stakeholders expressed a range of views on the suitability of the use of academic proficiency tests (such as IELTS) as university exit tests and for entry into the professions. With regard to IELTS, while some saw the relevance of the two writing tasks, particularly in relation to academic writing, others questioned the extent to which two timed tasks representing limited genres could elicit a representative sample of the professional writing required, particularly in the context of engineering. The findings are discussed in relation to different test purposes, the intersection between academic and specific purpose testing and the role of domain experts in test validation.

Genetic studies of the PRP17 gene of Saccharomyces cerevisiae: a domain essential for function maps to a nonconserved region of the protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the beta transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the beta transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between Prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is the N terminal region of the protein.

Lipid assisted membrane entry in specific phospholipid targeted protein systems

The purpose of this thesis project is to study changes in the physical state of cell membranes during cell entry, including how these changes are connected to the presence of ceramide. The role of enzymatical manipulation of lipids in bacterial internalization is also studied. A novel technique, where a single giant vesicle is chosen under the microscope and an enzyme coupled-particle attached to the micromanipulator pipette towards the vesicle, is used. Thus, the enzymatic reaction on the membrane of the giant vesicle can be followed in real-time. The first aim of this study is to develop a system where the localized sphingomyelinase membrane interaction could be observed on the surface of the giant vesicle and the effects could be monitored with microscopy. Domain formation, which resembles acid sphingomyelinase (ASMase), causes CD95 clustering in the cell membrane due to ceramide production (Grassmé et al., 2001a; Grassmé et al., 2001b) and the formation of small vesicles inside the manipulated giant vesicle is observed. Sphingomyelinase activation has also been found to be an important factor in the bacterial and viral invasion process in nonphagocytic cells (Grassmé et al., 1997; Jan et al., 2000). Accordingly, sphingomyelinase reactions in the cell membrane might also give insight into bacterial or viral cellular entry events. We found sphingomyelinase activity in Chlamydia pneumonia elementarybodies (EBs). Interestingly, the bacterium enters host cells by endocytosis but the internalization mechanism of Chlamydia is unknown. The hypothesis is that sphingomyelin is needed for host cell entry in the infection of C. pneumonia. The second project focuses on this subject. The goal of the third project is to study a role of phosphatidylserine as a target for a membrane binding protein. Phosphatidylserine is chosen because of its importance in fusion processes. This will be another example for the importance of lipids in cell targeting, internalization, and externalization.

Evolution of domain combinations in protein kinases and its implications for functional diversity

Protein kinases phosphorylating Ser/Thr/Tyr residues in several cellular proteins exert tight control over their biological functions. They constitute the largest protein family in most eukaryotic species. Protein kinases classified based on sequence similarity in their catalytic domains, cluster into subfamilies, which share gross functional properties. Many protein kinases are associated or tethered covalently to domains that serve as adapter or regulatory modules,naiding substrate recruitment, specificity, and also serve as scaffolds. Hence the modular organisation of the protein kinases serves as guidelines to their functional and molecular properties. Analysis of genomic repertoires of protein kinases in eukaryotes have revealed wide spectrum of domain organisation across various subfamilies of kinases. Occurrence of organism-specific novel domain combinations suggests functional diversity achieved by protein kinases in order to regulate variety of biological processes. In addition, domain architecture of protein kinases revealed existence of hybrid protein kinase subfamilies and their emerging roles in the signaling of eukaryotic organisms. In this review we discuss the repertoire of non-kinase domains tethered to multi-domain kinases in the metazoans. Similarities and differences in the domain architectures of protein kinases in these organisms indicate conserved and unique features that are critical to functional specialization. (C) 2009 Elsevier Ltd. All rights reserved.

Role of a PAS sensor domain in the Mycobacterium tuberculosis transcription regulator Rv1364c

The Mycobacterium tuberculosis transcriptional regulator Rv1364c regulates the activity of the stress response sigma factor sigma(F). This multi-domain protein has several components: a signaling PAS domain and an effector segment comprising of a phosphatase, a kinase and an anti-anti-sigma factor domain. Based on Small Angle X-ray Scattering (SAXS) data, Rv1364c was recently shown to be a homo-dimer and adopt an elongated conformation in solution. The PAS domain could not be modeled into the structural envelope due to poor sequence similarity with known PAS proteins. The crystal structure of the PAS domain described here provides a structural basis for the dimerization of Rv1364c. It thus appears likely that the PAS domain regulates the anti-sigma activity of Rv1364c by oligomerization. A structural comparison with other characterized PAS domains reveal several sequence and conformational features that could facilitate ligand binding - a feature which suggests that the function of Rv1364c could potentially be governed by specific cellular signals or metabolic cues. (C) 2010 Elsevier Inc. All rights reserved.

Crystal Structure of Staphylococcus aureus Metallopeptidase (Sapep) Reveals Large Domain Motions between the Manganese-bound and Apo-states

Proteases belonging to the M20 family are characterized by diverse substrate specificity and participate in several metabolic pathways. The Staphylococcus aureus metallopeptidase, Sapep, is a member of the aminoacylase-I/M20 protein family. This protein is a Mn2+-dependent dipeptidase. The crystal structure of this protein in the Mn2+-bound form and in the open, metal-free state suggests that large interdomain movements could potentially regulate the activity of this enzyme. We note that the extended inactive conformation is stabilized by a disulfide bond in the vicinity of the active site. Although these cysteines, Cys(155) and Cys(178), are not active site residues, the reduced form of this enzyme is substantially more active as a dipeptidase. These findings acquire further relevance given a recent observation that this enzyme is only active in methicillin-resistant S. aureus. The structural and biochemical features of this enzyme provide a template for the design of novel methicillin-resistant S. aureus-specific therapeutics.