DHODH modulates transcriptional elongation in the neural crest and melanoma

Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma1. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation

Site-directed mutagenesis reveals a unique requirement for tyrosine residues in IL-7Rα and TSLPR cytoplasmic domains in TSLP-dependent cell proliferation

<p>Abstract</p> <p>Background</p> <p>Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7) like cytokine, which plays an important role in the regulation of immune responses to allergens. TSLP binds to a heterodimeric receptor complex composed of the IL-7 receptor α chain (IL-7Rα) and the TSLP receptor (TSLPR, also known as CRLF2). It has previously been suggested that the lone tyrosine residue in the mouse TSLPR cytoplasmic domain is required for cell proliferation using chimeric receptor systems. Also the role of tyrosine residues in the IL-7Rα cytoplasmic domain in TSLP signaling has not yet been investigated. We undertook a systematic analysis to test the role of tyrosine residues of both the IL-7Rα and the TSLPR in inducing cell proliferation in a growth factor dependent cell line, Ba/F3.</p> <p>Results</p> <p>A multiple sequence alignment of the IL-7Rα and TSLPR cytoplasmic domains revealed conservation of most, but not all, cytoplasmic tyrosine residues across several species. Our site-directed mutagenesis experiments revealed that the single tyrosine residue in human TSLPR was not required for TSLP-dependent cell proliferation. It has previously been reported that Y449 of human IL-7Rα is required for IL-7 dependent proliferation. Interestingly, in contrast to IL-7 signaling, none of tyrosine residues in the human IL-7Rα cytoplasmic domain were required for TSLP-dependent cell proliferation in the presence of a wild type TSLPR. However, the mutation of all cytoplasmic four tyrosine residues of human IL-7Rα and human TSLPR to phenylalanine residues abolished the proliferative ability of the TSLP receptor complex in response to TSLP.</p> <p>Conclusion</p> <p>These results suggest that TSLP requires at least one cytoplasmic tyrosine residue to transmit proliferative signals. Unlike other members of IL-2 cytokine family, tyrosine residues in IL-7Rα and TSLPR cytoplasmic domains play a redundant role in TSLP-mediated cell growth.</p

Yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) as zoomonitors of environmental contamination at a polluted area in Slovakia

<p>Abstract</p> <p>Background</p> <p>Free-living wild rodents are often used as zoomonitors of environmental contamination. In the present study, accumulation of cadmium (Cd), copper (Cu), iron (Fe), and zinc (Zn) in critical organs of yellow-necked mice (<it>Apodemus flavicollis</it>) and bank voles (<it>Myodes glareolus</it>) trapped in a polluted area in Nováky, Slovakia was investigated.</p> <p>Methods</p> <p>Yellow-necked mice (n = 8) and bank voles (n = 10) were collected using standard theriological methods for wood ecosystems. All animals were adult males in good physical condition. The concentrations of Cd, Cu, Fe, and Zn in the liver, kidney, and bone were determined by atomic absorption spectrophotometry.</p> <p>Results</p> <p>The highest concentrations of Cd and Zn were found in the bone of both species while Cu and Fe accumulated mainly in kidney or liver. Significant higher concentrations of Cd and Cu were detected in the liver of bank voles than in yellow-necked mice. Similar significant higher levels of Cd and Zn were found in the bone of bank voles. In contrast, significant higher concentrations of Cu and Fe were present in the kidney of yellow-necked mice.</p> <p>Conclusions</p> <p>In the yellow-necked mouse and bank vole, bone seems to accumulate Cd and Zn following prolonged exposure. On the contrary, kidney and liver store Cu and Fe after a long-term environmental exposure. In the present study, bank voles seemed to be more heavy metal loaded zoomonitors than yellow-necked mice.</p

Editing independent effects of ADARs on the miRNA/siRNA pathways

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions

Preparation of Pre-Confluent Retinal Cells Increases Graft Viability In Vitro and In Vivo: A Mouse Model

PURPOSE: Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure "transplant conditions" (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01. METHODS: Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts. RESULTS: Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001). CONCLUSION: Pre-confluent cells should be used to maximize graft cell viability

Dimerisation induced formation of the active site and the identification of three metal sites in EAL-phosphodiesterases

The bacterial second messenger cyclic di-3′,5′-guanosine monophosphate (c-di-GMP) is a key regulator of bacterial motility and virulence. As high levels of c-di-GMP are associated with the biofilm lifestyle, c-di-GMP hydrolysing phosphodiesterases (PDEs) have been identified as key targets to aid development of novel strategies to treat chronic infection by exploiting biofilm dispersal. We have studied the EAL signature motif-containing phosphodiesterase domains from the Pseudomonas aeruginosa proteins PA3825 (PA3825EAL) and PA1727 (MucREAL). Different dimerisation interfaces allow us to identify interface independent principles of enzyme regulation. Unlike previously characterised two-metal binding EAL-phosphodiesterases, PA3825EAL in complex with pGpG provides a model for a third metal site. The third metal is positioned to stabilise the negative charge of the 5′-phosphate, and thus three metals could be required for catalysis in analogy to other nucleases. This newly uncovered variation in metal coordination may provide a further level of bacterial PDE regulation

Using data-driven rules to predict mortality in severe community acquired pneumonia

Prediction of patient-centered outcomes in hospitals is useful for performance benchmarking, resource allocation, and guidance regarding active treatment and withdrawal of care. Yet, their use by clinicians is limited by the complexity of available tools and amount of data required. We propose to use Disjunctive Normal Forms as a novel approach to predict hospital and 90-day mortality from instance-based patient data, comprising demographic, genetic, and physiologic information in a large cohort of patients admitted with severe community acquired pneumonia. We develop two algorithms to efficiently learn Disjunctive Normal Forms, which yield easy-to-interpret rules that explicitly map data to the outcome of interest. Disjunctive Normal Forms achieve higher prediction performance quality compared to a set of state-of-the-art machine learning models, and unveils insights unavailable with standard methods. Disjunctive Normal Forms constitute an intuitive set of prediction rules that could be easily implemented to predict outcomes and guide criteria-based clinical decision making and clinical trial execution, and thus of greater practical usefulness than currently available prediction tools. The Java implementation of the tool JavaDNF will be publicly available. © 2014 Wu et al

A Survey of Genomic Traces Reveals a Common Sequencing Error, RNA Editing, and DNA Editing

While it is widely held that an organism's genomic information should remain constant, several protein families are known to modify it. Members of the AID/APOBEC protein family can deaminate DNA. Similarly, members of the ADAR family can deaminate RNA. Characterizing the scope of these events is challenging. Here we use large genomic data sets, such as the two billion sequences in the NCBI Trace Archive, to look for clusters of mismatches of the same type, which are a hallmark of editing events caused by APOBEC3 and ADAR. We align 603,249,815 traces from the NCBI trace archive to their reference genomes. In clusters of mismatches of increasing size, at least one systematic sequencing error dominates the results (G-to-A). It is still present in mismatches with 99% accuracy and only vanishes in mismatches at 99.99% accuracy or higher. The error appears to have entered into about 1% of the HapMap, possibly affecting other users that rely on this resource. Further investigation, using stringent quality thresholds, uncovers thousands of mismatch clusters with no apparent defects in their chromatograms. These traces provide the first reported candidates of endogenous DNA editing in human, further elucidating RNA editing in human and mouse and also revealing, for the first time, extensive RNA editing in Xenopus tropicalis. We show that the NCBI Trace Archive provides a valuable resource for the investigation of the phenomena of DNA and RNA editing, as well as setting the stage for a comprehensive mapping of editing events in large-scale genomic datasets