Publications

Rapamycin reduces fibroblast proliferation without causing quiescence and induces STAT5A/B-mediated cytokine production

Posted on by biota in Publications

Gillespie ZE, MacKay K, Sander M, Trost B, Dawicki W, Wickramarathna A, Gordon J, Eramian M, Kill IR, Bridger JM, Kusalik A, Mitchell JA, Eskiw CH

Nucleus 2015;6(6):490-506

PMID: 26652669

Abstract

Rapamycin is a well-known inhibitor of the Target of Rapamycin (TOR) signaling cascade; however, the impact of this drug on global genome function and organization in normal primary cells is poorly understood. To explore this impact, we treated primary human foreskin fibroblasts with rapamycin and observed a decrease in cell proliferation without causing cell death. Upon rapamycin treatment chromosomes 18 and 10 were repositioned to a location similar to that of fibroblasts induced into quiescence by serum reduction. Although similar changes in positioning occurred, comparative transcriptome analyses demonstrated significant divergence in gene expression patterns between rapamycin-treated and quiescence-induced fibroblasts. Rapamycin treatment induced the upregulation of cytokine genes, including those from the Interleukin (IL)-6 signaling network, such as IL-8 and the Leukemia Inhibitory Factor (LIF), while quiescent fibroblasts demonstrated up-regulation of genes involved in the complement and coagulation cascade. In addition, genes significantly up-regulated by rapamycin treatment demonstrated increased promoter occupancy of the transcription factor Signal Transducer and Activator of Transcription 5A/B (STAT5A/B). In summary, we demonstrated that the treatment of fibroblasts with rapamycin decreased proliferation, caused chromosome territory repositioning and induced STAT5A/B-mediated changes in gene expression enriched for cytokines.

Elevated Temperature Differentially Influences Effector-Triggered Immunity Outputs in Arabidopsis

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Menna A, Nguyen D, Guttman DS, Desveaux D

Front Plant Sci 2015;6:995

PMID: 26617631

Abstract

Pseudomonas syringae is a Gram-negative bacterium that infects multiple plant species by manipulating cellular processes via injection of type three secreted effectors (T3SEs) into host cells. Nucleotide-binding leucine-rich repeat (NLR) resistance (R) proteins recognize specific T3SEs and trigger a robust immune response, called effector-triggered immunity (ETI), which limits pathogen proliferation and is often associated with localized programmed cell death, known as the hypersensitive response (HR). In this study, we examine the influence of elevated temperature on two ETI outputs: HR and pathogen virulence suppression. We found that in the Arabidopsis thaliana accession Col-0, elevated temperatures suppress the HR, but have minimal influence on ETI-associated P. syringae virulence suppression, thereby uncoupling these two ETI responses. We also identify accessions of Arabidopsis that exhibit impaired P. syringae virulence suppression at elevated temperature, highlighting the natural variation that exists in coping with biotic and abiotic stresses. These results not only reinforce the influence of abiotic factors on plant immunity but also emphasize the importance of carefully documented environmental conditions in studies of plant immunity.

Short linear motifs – ex nihilo evolution of protein regulation

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Davey NE, Cyert MS, Moses AM

Cell Commun. Signal 2015;13:43

PMID: 26589632

Abstract

Short sequence motifs are ubiquitous across the three major types of biomolecules: hundreds of classes and thousands of instances of DNA regulatory elements, RNA motifs and protein short linear motifs (SLiMs) have been characterised. The increase in complexity of transcriptional, post-transcriptional and post-translational regulation in higher Eukaryotes has coincided with a significant expansion of motif use. But how did the eukaryotic cell acquire such a vast repertoire of motifs? In this review, we curate the available literature on protein motif evolution and discuss the evidence that suggests SLiMs can be acquired by mutations, insertions and deletions in disordered regions. We propose a mechanism of ex nihilo SLiM evolution – the evolution of a novel SLiM from “nothing” – adding a functional module to a previously non-functional region of protein sequence. In our model, hundreds of motif-binding domains in higher eukaryotic proteins connect simple motif specificities with useful functions to create a large functional motif space. Accessible peptides that match the specificity of these motif-binding domains are continuously created and destroyed by mutations in rapidly evolving disordered regions, creating a dynamic supply of new interactions that may have advantageous phenotypic novelty. This provides a reservoir of diversity to modify existing interaction networks. Evolutionary pressures will act on these motifs to retain beneficial instances. However, most will be lost on an evolutionary timescale as negative selection and genetic drift act on deleterious and neutral motifs respectively. In light of the parallels between the presented model and the evolution of motifs in the regulatory segments of genes and (pre-)mRNAs, we suggest our understanding of regulatory networks would benefit from the creation of a shared model describing the evolution of transcriptional, post-transcriptional and post-translational regulation.

Global Analysis of the Fungal Microbiome in Cystic Fibrosis Patients Reveals Loss of Function of the Transcriptional Repressor Nrg1 as a Mechanism of Pathogen Adaptation

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Kim SH, Clark ST, Surendra A, Copeland JK, Wang PW, Ammar R, Collins C, Tullis DE, Nislow C, Hwang DM, Guttman DS, Cowen LE

PLoS Pathog. 2015 Nov;11(11):e1005308

PMID: 26588216

Abstract

The microbiome shapes diverse facets of human biology and disease, with the importance of fungi only beginning to be appreciated. Microbial communities infiltrate diverse anatomical sites as with the respiratory tract of healthy humans and those with diseases such as cystic fibrosis, where chronic colonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with Candida and Aspergillus species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within Candida species. Filamentation was uncoupled from inducing cues in 28 Candida isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of Pseudomonas aeruginosa, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.

The molecular origin and evolution of dim-light vision in mammals

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Bickelmann C, Morrow JM, Du J, Schott RK, van Hazel I, Lim S, Müller J, Chang BS

Evolution 2015 Nov;69(11):2995-3003

PMID: 26536060

Abstract

The nocturnal origin of mammals is a longstanding hypothesis that is considered instrumental for the evolution of endothermy, a potential key innovation in this successful clade. This hypothesis is primarily based on indirect anatomical inference from fossils. Here, we reconstruct the evolutionary history of rhodopsin–the vertebrate visual pigment mediating the first step in phototransduction at low-light levels–via codon-based model tests for selection, combined with gene resurrection methods that allow for the study of ancient proteins. Rhodopsin coding sequences were reconstructed for three key nodes: Amniota, Mammalia, and Theria. When expressed in vitro, all sequences generated stable visual pigments with λMAX values similar to the well-studied bovine rhodopsin. Retinal release rates of mammalian and therian ancestral rhodopsins, measured via fluorescence spectroscopy, were significantly slower than those of the amniote ancestor, indicating altered molecular function possibly related to nocturnality. Positive selection along the therian branch suggests adaptive evolution in rhodopsin concurrent with therian ecological diversification events during the Mesozoic that allowed for an exploration of the environment at varying light levels.

Short-term fertilizer application alters phenotypic traits of symbiotic nitrogen fixing bacteria

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Simonsen AK, Han S, Rekret P, Rentschler CS, Heath KD, Stinchcombe JR

PeerJ 2015;3:e1291

PMID: 26500812

Abstract

Fertilizer application is a common anthropogenic alteration to terrestrial systems. Increased nutrient input can impact soil microbial diversity or function directly through altered soil environments, or indirectly through plant-microbe feedbacks, with potentially important effects on ecologically-important plant-associated mutualists. We investigated the impacts of plant fertilizer, containing all common macro and micronutrients on symbiotic nitrogen-fixing bacteria (rhizobia), a group of bacteria that are important for plant productivity and ecosystem function. We collected rhizobia nodule isolates from natural field soil that was treated with slow-release plant fertilizer over a single growing season and compared phenotypic traits related to free-living growth and host partner quality in these isolates to those of rhizobia from unfertilized soils. Through a series of single inoculation assays in controlled glasshouse conditions, we found that isolates from fertilized field soil provided legume hosts with higher mutualistic benefits. Through growth assays on media containing variable plant fertilizer concentrations, we found that plant fertilizer was generally beneficial for rhizobia growth. Rhizobia isolated from fertilized field soil had higher growth rates in the presence of plant fertilizer compared to isolates from unfertilized field soil, indicating that plant fertilizer application favoured rhizobia isolates with higher abilities to utilize fertilizer for free-living growth. We found a positive correlation between growth responses to fertilizer and mutualism benefits among isolates from fertilized field soil, demonstrating that variable plant fertilizer induces context-dependent genetic correlations, potentially changing the evolutionary trajectory of either trait through increased trait dependencies. Our study shows that short-term application is sufficient to alter the composition of rhizobia isolates in the population or community, either directly though changes in the soil chemistry or indirectly through altered host legume feedbacks, and is potentially a strong selective agent acting on natural rhizobia populations.

Chronic infection phenotypes of Pseudomonas aeruginosa are associated with failure of eradication in children with cystic fibrosis

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Vidya P, Smith L, Beaudoin T, Yau YC, Clark S, Coburn B, Guttman DS, Hwang DM, Waters V

Eur. J. Clin. Microbiol. Infect. Dis. 2016 Jan;35(1):67-74

PMID: 26492874

Abstract

Early eradication treatment with inhaled tobramycin is successful in the majority of children with cystic fibrosis (CF) with incident Pseudomonas aeruginosa infection. However, in 10-40 % of cases, eradication fails and the reasons for this are poorly understood. The purpose of this study was to determine whether specific microbial characteristics could explain eradication treatment failure. This was a cross-sectional study of CF patients (aged 0-18 years) with incident P. aeruginosa infection from 2011 to 2014 at the Hospital for Sick Children, Toronto, Canada. Phenotypic assays were done on all incident P. aeruginosa isolates, and eradicated and persistent isolates were compared using the Mann-Whitney test or the two-sided Chi-square test. A total of 46 children with CF had 51 incident P. aeruginosa infections. In 72 % (33/46) of the patients, eradication treatment was successful, while 28 % failed eradication therapy. Persistent isolates were less likely to be motile, with significantly less twitch motility (p=0.001), were more likely to be mucoid (p=0.002), and more likely to have a tobramycin minimum inhibitory concentration (MIC) ≥ 128 μg/mL (p=0.02) compared to eradicated isolates. Although biofilm production was similar, there was a trend towards more persistent isolates with deletions in quorum-sensing genes compared with eradicated isolates (p=0.06). Initial acquisition of P. aeruginosa with characteristics of chronic infection is associated with failure of eradication treatment.

Spectral Tuning of Killer Whale (Orcinus orca) Rhodopsin: Evidence for Positive Selection and Functional Adaptation in a Cetacean Visual Pigment

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Dungan SZ, Kosyakov A, Chang BS

Mol. Biol. Evol. 2016 Feb;33(2):323-36

PMID: 26486871

Abstract

Cetaceans have undergone a remarkable evolutionary transition that was accompanied by many sensory adaptations, including modification of the visual system for underwater environments. Recent sequencing of cetacean genomes has made it possible to begin exploring the molecular basis of these adaptations. In this study we use in vitro expression methods to experimentally characterize the first step of the visual transduction cascade, the light activation of rhodopsin, for the killer whale. To investigate the spectral effects of amino acid substitutions thought to correspond with absorbance shifts relative to terrestrial mammals, we used the orca gene as a background for the first site-directed mutagenesis experiments in a cetacean rhodopsin. The S292A mutation had the largest effect, and was responsible for the majority of the spectral difference between killer whale and bovine (terrestrial) rhodopsin. Using codon-based likelihood models, we also found significant evidence for positive selection in cetacean rhodopsin sequences, including on spectral tuning sites we experimentally mutated. We then investigated patterns of ecological divergence that may be correlated with rhodopsin functional variation by using a series of clade models that partitioned the data set according to phylogeny, habitat, and foraging depth zone. Only the model partitioning according to depth was significant. This suggests that foraging dives might be a selective regime influencing cetacean rhodopsin divergence, and our experimental results indicate that spectral tuning may be playing an adaptive role in this process. Our study demonstrates that combining computational and experimental methods is crucial for gaining insight into the selection pressures underlying molecular evolution.

Concordance between RNA-sequencing data and DNA microarray data in transcriptome analysis of proliferative and quiescent fibroblasts

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Trost B, Moir CA, Gillespie ZE, Kusalik A, Mitchell JA, Eskiw CH

R Soc Open Sci 2015 Sep;2(9):150402

PMID: 26473061

Abstract

DNA microarrays and RNA sequencing (RNA-seq) are major technologies for performing high-throughput analysis of transcript abundance. Recently, concerns have been raised regarding the concordance of data derived from the two techniques. Using cDNA libraries derived from normal human foreskin fibroblasts, we measured changes in transcript abundance as cells transitioned from proliferative growth to quiescence using both DNA microarrays and RNA-seq. The internal reproducibility of the RNA-seq data was greater than that of the microarray data. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarray values were moderate. The two technologies had good agreement when considering probes with the largest (both positive and negative) fold change (FC) values. An independent technique, quantitative reverse-transcription PCR (qRT-PCR), was used to measure the FC of 76 genes between proliferative and quiescent samples, and a higher correlation was observed between the qRT-PCR data and the RNA-seq data than between the qRT-PCR data and the microarray data.

50 years of Arabidopsis research: highlights and future directions

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Provart NJ, Alonso J, Assmann SM, Bergmann D, Brady SM, Brkljacic J, Browse J, Chapple C, Colot V, Cutler S, Dangl J, Ehrhardt D, Friesner JD, Frommer WB, Grotewold E, Meyerowitz E, Nemhauser J, Nordborg M, Pikaard C, Shanklin J, Somerville C, Stitt M, Torii KU, Waese J, Wagner D, McCourt P

New Phytol. 2016 Feb;209(3):921-44

PMID: 26465351

Abstract

922 I. 922 II. 922 III. 925 IV. 925 V. 926 VI. 927 VII. 928 VIII. 929 IX. 930 X. 931 XI. 932 XII. 933 XIII. Natural variation and genome-wide association studies 934 XIV. 934 XV. 935 XVI. 936 XVII. 937 937 References 937 SUMMARY: The year 2014 marked the 25(th) International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. We present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.