Creating web applications for spatial epidemiological analysis and mapping in R using Rwui.

BACKGROUND: Creating a user friendly web based application which executes an R script allows physicians, epidemiologists, and others unfamiliar with the statistical language to perform powerful statistical analyses easily. The geographic mapping of data is an important tool in spatial epidemiological analysis, and the R project includes many tools for such analyses, but few for visualization. Hence, web applications that run R for epidemiological analysis need to be able to present the results in a geographic format. RESULTS: Rwui is a web application for creating web based applications for running R scripts. We describe updates to Rwui that enable it to create web applications for R scripts which return the results of the analysis to the web page as geographic maps. CONCLUSIONS: Rwui enables statisticians to create web applications for R scripts without the need to learn web programming. Creating a web application provides users access to an R based analysis without the need to learn R. Recent updates to Rwui have increased its applicability in the field of spatial epidemiological analysis.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Displaying R spatial statistics on Google dynamic maps with web applications created by Rwui.

BACKGROUND: The R project includes a large variety of packages designed for spatial statistics. Google dynamic maps provide web based access to global maps and satellite imagery. We describe a method for displaying directly the spatial output from an R script on to a Google dynamic map. METHODS: This is achieved by creating a Java based web application which runs the R script and then displays the results on the dynamic map. In order to make this method easy to implement by those unfamiliar with programming Java based web applications, we have added the method to the options available in the R Web User Interface (Rwui) application. Rwui is an established web application for creating web applications for running R scripts. A feature of Rwui is that all the code for the web application being created is generated automatically so that someone with no knowledge of web programming can make a fully functional web application for running an R script in a matter of minutes. RESULTS: Rwui can now be used to create web applications that will display the results from an R script on a Google dynamic map. Results may be displayed as discrete markers and/or as continuous overlays. In addition, users of the web application may select regions of interest on the dynamic map with mouse clicks and the coordinates of the region of interest will automatically be made available for use by the R script. CONCLUSIONS: This method of displaying R output on dynamic maps is designed to be of use in a number of areas. Firstly it allows statisticians, working in R and developing methods in spatial statistics, to easily visualise the results of applying their methods to real world data. Secondly, it allows researchers who are using R to study health geographics data, to display their results directly onto dynamic maps. Thirdly, by creating a web application for running an R script, a statistician can enable users entirely unfamiliar with R to run R coded statistical analyses of health geographics data. Fourthly, we envisage an educational role for such applications.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

An atlas of mouse CD4(+) T cell transcriptomes.

BACKGROUND: CD4(+) T cells are key regulators of the adaptive immune system and can be divided into T helper (Th) cells and regulatory T (Treg) cells. During an immune response Th cells mature from a naive state into one of several effector subtypes that exhibit distinct functions. The transcriptional mechanisms that underlie the specific functional identity of CD4(+) T cells are not fully understood. RESULTS: To assist investigations into the transcriptional identity and regulatory processes of these cells we performed mRNA-sequencing on three murine T helper subtypes (Th1, Th2 and Th17) as well as on splenic Treg cells and induced Treg (iTreg) cells. Our integrated analysis of this dataset revealed the gene expression changes associated with these related but distinct cellular identities. Each cell subtype differentially expresses a wealth of 'subtype upregulated' genes, some of which are well known whilst others promise new insights into signalling processes and transcriptional regulation. We show that hundreds of genes are regulated purely by alternative splicing to extend our knowledge of the role of post-transcriptional regulation in cell differentiation. CONCLUSIONS: This CD4(+) transcriptome atlas provides a valuable resource for the study of CD4(+) T cell populations. To facilitate its use by others, we have made the data available in an easily accessible online resource at www.th-express.org

Depletion of stromal cells expressing fibroblast activation protein-α from skeletal muscle and bone marrow results in cachexia and anemia.

Fibroblast activation protein-α (FAP) identifies stromal cells of mesenchymal origin in human cancers and chronic inflammatory lesions. In mouse models of cancer, they have been shown to be immune suppressive, but studies of their occurrence and function in normal tissues have been limited. With a transgenic mouse line permitting the bioluminescent imaging of FAP(+) cells, we find that they reside in most tissues of the adult mouse. FAP(+) cells from three sites, skeletal muscle, adipose tissue, and pancreas, have highly similar transcriptomes, suggesting a shared lineage. FAP(+) cells of skeletal muscle are the major local source of follistatin, and in bone marrow they express Cxcl12 and KitL. Experimental ablation of these cells causes loss of muscle mass and a reduction of B-lymphopoiesis and erythropoiesis, revealing their essential functions in maintaining normal muscle mass and hematopoiesis, respectively. Remarkably, these cells are altered at these sites in transplantable and spontaneous mouse models of cancer-induced cachexia and anemia. Thus, the FAP(+) stromal cell may have roles in two adverse consequences of cancer: their acquisition by tumors may cause failure of immunosurveillance, and their alteration in normal tissues contributes to the paraneoplastic syndromes of cachexia and anemia

Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought about by an environmental stressor, demonstrate important differences in aging- versus stress-induced protein damage, and challenge the widely held view that chemical chaperones are accumulated during hypertonic stress to protect protein structure/function

The elucidation of immunological and oncological transcriptomic signatures using translational ontologies and next-generation sequencing

Understanding the large amounts of information produced by next-generation sequencing requires the comprehensive integration of biological knowledge, appropriate statistical frameworks, and computational models. In this work, a series of computational approaches and analyses are presented which outline a path from raw data to an understanding of biological regulation, disease pathogenesis, and mutational behaviour. The first chapter, “The Bioinformatics of Next Generation Sequencing” consists of the biological and computational background to next-generation sequencing. The next chapter, called “Ontology-Driven Investigation of the Immunome and Diseasome” consists of a description of the biomedical ontology with immunological functional annotations, and comprehensive disease-gene relationships. In chapter 3, “An Ontology-based Pipeline for Transcriptomic Analysis,” the computational approaches to next-generation sequencing will be integrated with the ontologies created in chapter 2. Here, a novel pipeline will be presented which encapsulates quality control, normalization, quantification, differential expression analysis, and functional annotation. This pipeline was tested with a set of transcriptomic data and corresponding proteomic data, facilitating the exploration of the relationship between these two datasets. Chapter 4, “Transcriptomic Signatures in Oncology and Immunology,” consists of two parts. First, a novel ontology-based barcoding algorithm was developed, and a murine pancreatic cancer model was analyzed to identify key pancreatic cancer genes. A pharmaceutical treatment candidate was also identified and biologically validated in a mouse model. In the second part of the chapter, a similar method was used to understand the role of Foxp3 architecture in immune system biology. These methods will then be framed in the context of broader biological and clinical applications. From these oncological and immunological analyses, a “central dogma” of transcriptomic expression patterns can be derived. In chapter 5, key conclusions and future directions will be discussed in more detail, and the translational implications of the pipeline and ontology explored

Regulation of the Heat Shock Response and HSF-1 Nuclear Stress Bodies in C. elegans

The Heat Shock Response (HSR) is a highly conserved stress responsive molecular pathway that functions to promote appropriate protein folding in the cell. The HSR accomplishes this primarily through the use of molecular chaperones that serve to bind to misfolded or unfolded proteins to assist in stabilizing and folding proteins back to their native functional state. The master regulator of this pathway is a transcription factor known as Heat Shock Factor 1 (HSF1). HSF1 regulates molecular chaperone expression in the cell’s basal state, but can also be stress induced by diverse biotic and abiotic signals including thermal shock, oxidative stress, osmotic imbalance, pathogenic invasion, cell transformation, and other pathological disease states. Thus, it is essential to understand how HSF1 function is regulated to better appreciate how the compromise of protein homeostasis (proteostasis) underlies many clinical disease pathologies. Evidence from invertebrates suggests that the HSR undergoes a rapid decline very early in adulthood and may explain the physiological effect of aging across many cell and tissue types. To better understand this process, we sought to develop an endogenously tagged fluorescent model of HSF1 in C. elegans. We utilized CRISPR/Cas9 mediated transgenesis and found that our tagged model behaves very similar to wildtype animals and displays similar phenotypes to previously published low-copy fluorescent models of HSF-1 which is the C. elegans homolog of mammalian HSF1. Using this novel model, we find that HSF-1 is capable of responding to novel cell stressors including Juglone, Peroxide, Paraquat, Osmotic stress, and UV exposure. This model also displays tissue-specific localization changes during the transition to adulthood. This time period of around 24 hours has been shown to be the critical window where the HSR collapses. The formation of these age-related HSF-1::GFP nuclear stress bodies (nSBs) is typically correlated with an increase in HSR activity, yet multiple measurements of proteostasis in the worm suggest that HSF-1 cannot mount an adequate response to meet acute stress demands. Genetic loss of the germline that has been shown previously to enhance longevity and stress resistance was able to suppress the formation of nSBs suggesting that the HSR remains robust and retains the youthful phenotype. Our data suggests that most cells in the worm form HSF-1:GFP nSBs during this early timepoint except the neurons. We hypothesized that this may be due to physical contact and examined the effect of ensheathment defective mutants, but found no difference in the appearance of nSBs after the transition to adulthood. Recent data in the literature suggested that chromatin remodel may underlie the abrupt decline in the HSR has previously stated. To identify other candidate chromatin remodeling genes we performed a targeted RNAi subscreen to search for other regulators of the HSR across the transition to adulthood. Our work identified pyp-1, an inorganic pyrophosphatase, that when suppressed is capable of enhancing the activity of HSR transcriptional reporters and can also support metastable protein folding reporter animals. Interestingly, we did not find a subsequent benefit in longevity due to this increased HSF-1 dependent activity. Additionally, the effect of pyp-1 knockdown on our reporter animals appeared to require initiation of RNAi prior to the transition of adulthood. Taken together, this data may suggest that pyp-1 performs a specific function during the transition to adulthood and that when this process is suppressed it results in increased HSF-1 activity in adulthood, but it is not sufficient to more broadly enhance proteostasis. This suggests further investigation into pyp-1 expression and activity to better understand its role in regulating the HSR. The literature suggests that mammalian HSF1 can be post-translationally modified by O-GlcNAclyation. This modification, which is similar to phosphorylation, is thought to be very dynamic and highly dysregulated in many metabolic disorders including diabetes, cancer, and neurodegeneration. The overall effect of O-GlcNAclyation on the HSR at the organismal level is still unknown. To investigate the role of O-GlcNAclyation in C. elegans, we utilized knockdown of the two O-GlcNAclyation modifying enzymes, oga-1 and ogt-1, to examine the effect of hyper-O-GlcNAclyation and hypo-O-GlcNAclyation on the HSR in the worm. We found that in larval animals disruption O-GlcNAc cycling typically results in the enhancement of proteostasis. However, in adults, we found that knockdown of oga-1 typically resulted in increased HSF-1 activity and ogt-1 knockdown compromised proteostasis. Interestingly, we found that modulating O-GlcNAc cycling appeared to alter HSF-1::GFP localization specifically in the intestine suggesting further research. Intriguingly, when performing experiments to confirm the modulation of O-GlcNAc cycling on the HSR was HSF-1 dependent we found a dramatic reversal of the phenotypes of oga-1 and ogt-1 genetic dosage. This conflict may suggest that the bacterial food source, RNAi pathway activation, or other factors may synergize with O-GlcNAclyation to specifically regulate the HSR and suggests future experimentation. Previous research from our lab suggests that HSF-1 may regulate a number of collagen and cuticle genes in C. elegans both in basal conditions and during acute stress. It was suggested that these collagen and cuticle genes may themselves regulate the HSR. To address this, we performed a RNAi subscreen of all available cuticle and collagen genes using a hsf-16.2 fluorescent transcriptional reporter. We found a number of candidate genes that both enhanced and suppressed stress induction relative to control knockdown. Further research is required to determine if these candidates also regulate endogenous HSF-1 target gene expression and by what mechanism this is performed with. Lastly, we utilized our validated HSF-1::GFP CRISPR/Cas9 model to examine the genetic regulation of HSF-1:GFP nSBs. It has been shown that the formation of HSF1 nSBs are typically correlated with an increase in HSR activity, but prolonged HSF1 nSBs is associated with a compromise in proteostasis. Similar to our previous research we find that longevity enhancing genetic backgrounds typically suppress HSF-1::GFP nSB formation during the transition to adulthood. Previously, we found that genetic loss of the germline conferred by a glp-1 mutation blocked the formation of these nSBs. Here we found that this effect requires p38 MAPK signaling as a pmk-1 mutant in the glp-1 mutant background reversed the effect of glp-1. Next, we found that SIR-2.1 overexpression also suppressed nSBs that form during the transition to adulthood and that this required the lysine demethylase jmjd-3.1. We also examined the role of disrupting insulin signaling which is well known to dramatically enhance longevity and stress resistance. Interestingly, we did find less nSBs relative to wildtype but the effect was not completely suppressed as seen in glp-1 and SIR-2.1 OE genetic backgrounds. Finally, we examined the role of hsb-1 in regulating HSF-1::GFP nSBs and found that hsb-1 mutants typically had increased nSBs and a delay in restoring the basal level of nSBs after acute stress. Also, in the hsb-1 mutant background, we found that jmjd-3.1 expression is enhanced which has been previously shown to regulate HSF-1’s chromatin accessibility to its target hsps. Taken together, this entire work establishes an endogenously tagged whole-organism model of HSF-1 and expands upon the knowledge of stress conditions that regulate HSF-1. We also identify novel genetic pathways at the whole organism level to regulate the HSR including age-specific modulation of inorganic pyrophosphatase, post-translation modification pathways, and manipulation to the worm cuticle. These identified signaling cascades require further research work to fully understand how each contributes to HSF-1 regulation and in what tissue types this regulation is present in

Regulation of the Heat Shock Response and HSF-1 Nuclear Stress Bodies in C. elegans

The Heat Shock Response (HSR) is a highly conserved stress responsive molecular pathway that functions to promote appropriate protein folding in the cell. The HSR accomplishes this primarily through the use of molecular chaperones that serve to bind to misfolded or unfolded proteins to assist in stabilizing and folding proteins back to their native functional state. The master regulator of this pathway is a transcription factor known as Heat Shock Factor 1 (HSF1). HSF1 regulates molecular chaperone expression in the cell’s basal state, but can also be stress induced by diverse biotic and abiotic signals including thermal shock, oxidative stress, osmotic imbalance, pathogenic invasion, cell transformation, and other pathological disease states. Thus, it is essential to understand how HSF1 function is regulated to better appreciate how the compromise of protein homeostasis (proteostasis) underlies many clinical disease pathologies. Evidence from invertebrates suggests that the HSR undergoes a rapid decline very early in adulthood and may explain the physiological effect of aging across many cell and tissue types. To better understand this process, we sought to develop an endogenously tagged fluorescent model of HSF1 in C. elegans. We utilized CRISPR/Cas9 mediated transgenesis and found that our tagged model behaves very similar to wildtype animals and displays similar phenotypes to previously published low-copy fluorescent models of HSF-1 which is the C. elegans homolog of mammalian HSF1. Using this novel model, we find that HSF-1 is capable of responding to novel cell stressors including Juglone, Peroxide, Paraquat, Osmotic stress, and UV exposure. This model also displays tissue-specific localization changes during the transition to adulthood. This time period of around 24 hours has been shown to be the critical window where the HSR collapses. The formation of these age-related HSF-1::GFP nuclear stress bodies (nSBs) is typically correlated with an increase in HSR activity, yet multiple measurements of proteostasis in the worm suggest that HSF-1 cannot mount an adequate response to meet acute stress demands. Genetic loss of the germline that has been shown previously to enhance longevity and stress resistance was able to suppress the formation of nSBs suggesting that the HSR remains robust and retains the youthful phenotype. Our data suggests that most cells in the worm form HSF-1:GFP nSBs during this early timepoint except the neurons. We hypothesized that this may be due to physical contact and examined the effect of ensheathment defective mutants, but found no difference in the appearance of nSBs after the transition to adulthood. Recent data in the literature suggested that chromatin remodel may underlie the abrupt decline in the HSR has previously stated. To identify other candidate chromatin remodeling genes we performed a targeted RNAi subscreen to search for other regulators of the HSR across the transition to adulthood. Our work identified pyp-1, an inorganic pyrophosphatase, that when suppressed is capable of enhancing the activity of HSR transcriptional reporters and can also support metastable protein folding reporter animals. Interestingly, we did not find a subsequent benefit in longevity due to this increased HSF-1 dependent activity. Additionally, the effect of pyp-1 knockdown on our reporter animals appeared to require initiation of RNAi prior to the transition of adulthood. Taken together, this data may suggest that pyp-1 performs a specific function during the transition to adulthood and that when this process is suppressed it results in increased HSF-1 activity in adulthood, but it is not sufficient to more broadly enhance proteostasis. This suggests further investigation into pyp-1 expression and activity to better understand its role in regulating the HSR. The literature suggests that mammalian HSF1 can be post-translationally modified by O-GlcNAclyation. This modification, which is similar to phosphorylation, is thought to be very dynamic and highly dysregulated in many metabolic disorders including diabetes, cancer, and neurodegeneration. The overall effect of O-GlcNAclyation on the HSR at the organismal level is still unknown. To investigate the role of O-GlcNAclyation in C. elegans, we utilized knockdown of the two O-GlcNAclyation modifying enzymes, oga-1 and ogt-1, to examine the effect of hyper-O-GlcNAclyation and hypo-O-GlcNAclyation on the HSR in the worm. We found that in larval animals disruption O-GlcNAc cycling typically results in the enhancement of proteostasis. However, in adults, we found that knockdown of oga-1 typically resulted in increased HSF-1 activity and ogt-1 knockdown compromised proteostasis. Interestingly, we found that modulating O-GlcNAc cycling appeared to alter HSF-1::GFP localization specifically in the intestine suggesting further research. Intriguingly, when performing experiments to confirm the modulation of O-GlcNAc cycling on the HSR was HSF-1 dependent we found a dramatic reversal of the phenotypes of oga-1 and ogt-1 genetic dosage. This conflict may suggest that the bacterial food source, RNAi pathway activation, or other factors may synergize with O-GlcNAclyation to specifically regulate the HSR and suggests future experimentation. Previous research from our lab suggests that HSF-1 may regulate a number of collagen and cuticle genes in C. elegans both in basal conditions and during acute stress. It was suggested that these collagen and cuticle genes may themselves regulate the HSR. To address this, we performed a RNAi subscreen of all available cuticle and collagen genes using a hsf-16.2 fluorescent transcriptional reporter. We found a number of candidate genes that both enhanced and suppressed stress induction relative to control knockdown. Further research is required to determine if these candidates also regulate endogenous HSF-1 target gene expression and by what mechanism this is performed with. Lastly, we utilized our validated HSF-1::GFP CRISPR/Cas9 model to examine the genetic regulation of HSF-1:GFP nSBs. It has been shown that the formation of HSF1 nSBs are typically correlated with an increase in HSR activity, but prolonged HSF1 nSBs is associated with a compromise in proteostasis. Similar to our previous research we find that longevity enhancing genetic backgrounds typically suppress HSF-1::GFP nSB formation during the transition to adulthood. Previously, we found that genetic loss of the germline conferred by a glp-1 mutation blocked the formation of these nSBs. Here we found that this effect requires p38 MAPK signaling as a pmk-1 mutant in the glp-1 mutant background reversed the effect of glp-1. Next, we found that SIR-2.1 overexpression also suppressed nSBs that form during the transition to adulthood and that this required the lysine demethylase jmjd-3.1. We also examined the role of disrupting insulin signaling which is well known to dramatically enhance longevity and stress resistance. Interestingly, we did find less nSBs relative to wildtype but the effect was not completely suppressed as seen in glp-1 and SIR-2.1 OE genetic backgrounds. Finally, we examined the role of hsb-1 in regulating HSF-1::GFP nSBs and found that hsb-1 mutants typically had increased nSBs and a delay in restoring the basal level of nSBs after acute stress. Also, in the hsb-1 mutant background, we found that jmjd-3.1 expression is enhanced which has been previously shown to regulate HSF-1’s chromatin accessibility to its target hsps. Taken together, this entire work establishes an endogenously tagged whole-organism model of HSF-1 and expands upon the knowledge of stress conditions that regulate HSF-1. We also identify novel genetic pathways at the whole organism level to regulate the HSR including age-specific modulation of inorganic pyrophosphatase, post-translation modification pathways, and manipulation to the worm cuticle. These identified signaling cascades require further research work to fully understand how each contributes to HSF-1 regulation and in what tissue types this regulation is present in