Transcriptome Profiling Reveals Enhanced Mitochondrial Activity as a Cold Adaptive Strategy to Hypothermia in Zebrafish Muscle.

The utilisation of synthetic torpor for interplanetary travel once seemed farfetched. However, mounting evidence points to torpor-induced protective benefits from the main hazards of space travel, namely, exposure to radiation and microgravity. To determine the radio-protective effects of an induced torpor-like state we exploited the ectothermic nature of the Danio rerio (zebrafish) in reducing their body temperatures to replicate the hypothermic states seen during natural torpor. We also administered melatonin as a sedative to reduce physical activity. Zebrafish were then exposed to low-dose radiation (0.3 Gy) to simulate radiation exposure on long-term space missions. Transcriptomic analysis found that radiation exposure led to an upregulation of inflammatory and immune signatures and a differentiation and regeneration phenotype driven by STAT3 and MYOD1 transcription factors. In addition, DNA repair processes were downregulated in the muscle two days' post-irradiation. The effects of hypothermia led to an increase in mitochondrial translation including genes involved in oxidative phosphorylation and a downregulation of extracellular matrix and developmental genes. Upon radiation exposure, increases in endoplasmic reticulum stress genes were observed in a torpor+radiation group with downregulation of immune-related and ECM genes. Exposing hypothermic zebrafish to radiation also resulted in a downregulation of ECM and developmental genes however, immune/inflammatory related pathways were downregulated in contrast to that observed in the radiation only group. A cross-species comparison was performed with the muscle of hibernating Ursus arctos horribilis (brown bear) to define shared mechanisms of cold tolerance. Shared responses show an upregulation of protein translation and metabolism of amino acids, as well as a hypoxia response with the shared downregulation of glycolysis, ECM, and developmental genes.

Validation of In Vitro Trained Transcriptomic Radiosensitivity Signatures in Clinical Cohorts.

Transcriptomic personalisation of radiation therapy has gained considerable interest in recent years. However, independent model testing on in vitro data has shown poor performance. In this work, we assess the reproducibility in clinical applications of radiosensitivity signatures. Agreement between radiosensitivity predictions from published signatures using different microarray normalization methods was assessed. Control signatures developed from resampled in vitro data were benchmarked in clinical cohorts. Survival analysis was performed using each gene in the clinical transcriptomic data, and gene set enrichment analysis was used to determine pathways related to model performance in predicting survival and recurrence. The normalisation approach impacted calculated radiosensitivity index (RSI) values. Indeed, the limits of agreement exceeded 20% with different normalisation approaches. No published signature significantly improved on the resampled controls for prediction of clinical outcomes. Functional annotation of gene models suggested that many overlapping biological processes are associated with cancer outcomes in RT treated and non-RT treated patients, including proliferation and immune responses. In summary, different normalisation methods should not be used interchangeably. The utility of published signatures remains unclear given the large proportion of genes relating to cancer outcome. Biological processes influencing outcome overlapped for patients treated with or without radiation suggest that existing signatures may lack specificity.

Investigating the effects of chronic low-dose radiation exposure in the liver of a hypothermic zebrafish model.

Mankind's quest for a manned mission to Mars is placing increased emphasis on the development of innovative radio-protective countermeasures for long-term space travel. Hibernation confers radio-protective effects in hibernating animals, and this has led to the investigation of synthetic torpor to mitigate the deleterious effects of chronic low-dose-rate radiation exposure. Here we describe an induced torpor model we developed using the zebrafish. We explored the effects of radiation exposure on this model with a focus on the liver. Transcriptomic and behavioural analyses were performed. Radiation exposure resulted in transcriptomic perturbations in lipid metabolism and absorption, wound healing, immune response, and fibrogenic pathways. Induced torpor reduced metabolism and increased pro-survival, anti-apoptotic, and DNA repair pathways. Coupled with radiation exposure, induced torpor led to a stress response but also revealed maintenance of DNA repair mechanisms, pro-survival and anti-apoptotic signals. To further characterise our model of induced torpor, the zebrafish model was compared with hepatic transcriptomic data from hibernating grizzly bears (Ursus arctos horribilis) and active controls revealing conserved responses in gene expression associated with anti-apoptotic processes, DNA damage repair, cell survival, proliferation, and antioxidant response. Similarly, the radiation group was compared with space-flown mice revealing shared changes in lipid metabolism.

PTEN Protein Phosphatase Activity Is Not Required for Tumour Suppression in the Mouse Prostate.

Loss PTEN function is one of the most common events driving aggressive prostate cancers and biochemically, PTEN is a lipid phosphatase which opposes the activation of the oncogenic PI3K-AKT signalling network. However, PTEN also has additional potential mechanisms of action, including protein phosphatase activity. Using a mutant enzyme, PTEN Y138L, which selectively lacks protein phosphatase activity, we characterised genetically modified mice lacking either the full function of PTEN in the prostate gland or only lacking protein phosphatase activity. The phenotypes of mice carrying a single allele of either wild-type Pten or PtenY138L in the prostate were similar, with common prostatic intraepithelial neoplasia (PIN) and similar gene expression profiles. However, the latter group, lacking PTEN protein phosphatase activity additionally showed lymphocyte infiltration around PIN and an increased immune cell gene expression signature. Prostate adenocarcinoma, elevated proliferation and AKT activation were only frequently observed when PTEN was fully deleted. We also identify a common gene expression signature of PTEN loss conserved in other studies (including Nkx3.1, Tnf and Cd44). We provide further insight into tumour development in the prostate driven by loss of PTEN function and show that PTEN protein phosphatase activity is not required for tumour suppression.

Immune Cell Networks Uncover Candidate Biomarkers of Melanoma Immunotherapy Response.

The therapeutic activation of antitumour immunity by immune checkpoint inhibitors (ICIs) is a significant advance in cancer medicine, not least due to the prospect of long-term remission. However, many patients are unresponsive to ICI therapy and may experience serious side effects; companion biomarkers are urgently needed to help inform ICI prescribing decisions. We present the IMMUNETS networks of gene coregulation in five key immune cell types and their application to interrogate control of nivolumab response in advanced melanoma cohorts. The results evidence a role for each of the IMMUNETS cell types in ICI response and in driving tumour clearance with independent cohorts from TCGA. As expected, 'immune hot' status, including T cell proliferation, correlates with response to first-line ICI therapy. Genes regulated in NK, dendritic, and B cells are the most prominent discriminators of nivolumab response in patients that had previously progressed on another ICI. Multivariate analysis controlling for tumour stage and age highlights CIITA and IKZF3 as candidate prognostic biomarkers. IMMUNETS provide a resource for network biology, enabling context-specific analysis of immune components in orthogonal datasets. Overall, our results illuminate the relationship between the tumour microenvironment and clinical trajectories, with potential implications for precision medicine.

RadSigBench: a framework for benchmarking functional genomics signatures of cancer cell radiosensitivity.

Multiple transcriptomic predictors of tumour cell radiosensitivity (RS) have been proposed, but they have not been benchmarked against one another or to control models. To address this, we present RadSigBench, a comprehensive benchmarking framework for RS signatures. The approach compares candidate models to those developed from randomly resampled control signatures and from cellular processes integral to the radiation response. Robust evaluation of signature accuracy, both overall and for individual tissues, is performed. The NCI60 and Cancer Cell Line Encyclopaedia datasets are integrated into our workflow. Prediction of two measures of RS is assessed: survival fraction after 2 Gy and mean inactivation dose. We apply the RadSigBench framework to seven prominent published signatures of radiation sensitivity and test for equivalence to control signatures. The mean out-of-sample R2 for the published models on test data was very poor at 0.01 (range: -0.05 to 0.09) for Cancer Cell Line Encyclopedia and 0.00 (range: -0.19 to 0.19) in the NCI60 data. The accuracy of both published and cellular process signatures investigated was equivalent to the resampled controls, suggesting that these signatures contain limited radiation-specific information. Enhanced modelling strategies are needed for effective prediction of intrinsic RS to inform clinical treatment regimes. We make recommendations for methodological improvements, for example the inclusion of perturbation data, multiomics, advanced machine learning and mechanistic modelling. Our validation framework provides for robust performance assessment of ongoing developments in intrinsic RS prediction.

Strategies and techniques for quality control and semantic enrichment with multimodal data: a case study in colorectal cancer with eHDPrep.

BACKGROUND: Integration of data from multiple domains can greatly enhance the quality and applicability of knowledge generated in analysis workflows. However, working with health data is challenging, requiring careful preparation in order to support meaningful interpretation and robust results. Ontologies encapsulate relationships between variables that can enrich the semantic content of health datasets to enhance interpretability and inform downstream analyses. FINDINGS: We developed an R package for electronic health data preparation, "eHDPrep," demonstrated upon a multimodal colorectal cancer dataset (661 patients, 155 variables; Colo-661); a further demonstrator is taken from The Cancer Genome Atlas (459 patients, 94 variables; TCGA-COAD). eHDPrep offers user-friendly methods for quality control, including internal consistency checking and redundancy removal with information-theoretic variable merging. Semantic enrichment functionality is provided, enabling generation of new informative "meta-variables" according to ontological common ancestry between variables, demonstrated with SNOMED CT and the Gene Ontology in the current study. eHDPrep also facilitates numerical encoding, variable extraction from free text, completeness analysis, and user review of modifications to the dataset. CONCLUSIONS: eHDPrep provides effective tools to assess and enhance data quality, laying the foundation for robust performance and interpretability in downstream analyses. Application to multimodal colorectal cancer datasets resulted in improved data quality, structuring, and robust encoding, as well as enhanced semantic information. We make eHDPrep available as an R package from CRAN (https://cran.r-project.org/package = eHDPrep) and GitHub (https://github.com/overton-group/eHDPrep).

Long non-coding RNAs and their potential impact on diagnosis, prognosis, and therapy in prostate cancer: racial, ethnic, and geographical considerations.

INTRODUCTION: Advances in high-throughput sequencing have greatly advanced our understanding of long non-coding RNAs (lncRNAs) in a relatively short period of time. This has expanded our knowledge of cancer, particularly how lncRNAs drive many important cancer phenotypes via their regulation of gene expression. AREAS COVERED: Men of African descent are disproportionately affected by PC in terms of incidence, morbidity, and mortality. LncRNAs could serve as biomarkers to differentiate low-risk from high-risk diseases. Additionally, they may represent therapeutic targets for advanced and castrate-resistant cancer. We review current research surrounding lncRNAs and their association with PC. We discuss how lncRNAs can provide new insights and diagnostic biomarkers for African American men. Finally, we review advances in computational approaches that predict the regulatory effects of lncRNAs in cancer. EXPERT OPINION: PC diagnostic biomarkers that offer high specificity and sensitivity are urgently needed. PC specific lncRNAs are compelling as diagnostic biomarkers owing to their high tissue and tumor specificity and presence in bodily fluids. Recent studies indicate that PCA3 clinical utility might be restricted to men of European descent. Further work is required to develop lncRNA biomarkers tailored for men of African descent.

SynLeGG: analysis and visualization of multiomics data for discovery of cancer 'Achilles Heels' and gene function relationships.

Achilles' heel relationships arise when the status of one gene exposes a cell's vulnerability to perturbation of a second gene, such as chemical inhibition, providing therapeutic opportunities for precision oncology. SynLeGG (www.overton-lab.uk/synlegg) identifies and visualizes mutually exclusive loss signatures in 'omics data to enable discovery of genetic dependency relationships (GDRs) across 783 cancer cell lines and 30 tissues. While there is significant focus on genetic approaches, transcriptome data has advantages for investigation of GDRs and remains relatively underexplored. SynLeGG depends upon the MultiSEp algorithm for unsupervised assignment of cell lines into gene expression clusters, which provide the basis for analysis of CRISPR scores and mutational status in order to propose candidate GDRs. Benchmarking against SynLethDB demonstrates favourable performance for MultiSEp against competing approaches, finding significantly higher area under the Receiver Operator Characteristic curve and between 2.8-fold to 8.5-fold greater coverage. In addition to pan-cancer analysis, SynLeGG offers investigation of tissue-specific GDRs and recovers established relationships, including synthetic lethality for SMARCA2 with SMARCA4. Proteomics, Gene Ontology, protein-protein interactions and paralogue information are provided to assist interpretation and candidate drug target prioritization. SynLeGG predictions are significantly enriched in dependencies validated by a recently published CRISPR screen.

Induced Torpor as a Countermeasure for Low Dose Radiation Exposure in a Zebrafish Model.

The development of the Artemis programme with the goal of returning to the moon is spurring technology advances that will eventually take humans to Mars and herald a new era of interplanetary space travel. However, long-term space travel poses unique challenges including exposure to ionising radiation from galactic cosmic rays and potential solar particle events, exposure to microgravity and specific nutritional challenges arising from earth independent exploration. Ionising radiation is one of the major obstacles facing future space travel as it can generate oxidative stress and directly damage cellular structures such as DNA, in turn causing genomic instability, telomere shortening, extracellular-matrix remodelling and persistent inflammation. In the gastrointestinal tract (GIT) this can lead to leaky gut syndrome, perforations and motility issues, which impact GIT functionality and affect nutritional status. While current countermeasures such as shielding from the spacecraft can attenuate harmful biological effects, they produce harmful secondary particles that contribute to radiation exposure. We hypothesised that induction of a torpor-like state would confer a radioprotective effect given the evidence that hibernation extends survival times in irradiated squirrels compared to active controls. To test this hypothesis, a torpor-like state was induced in zebrafish using melatonin treatment and reduced temperature, and radiation exposure was administered twice over the course of 10 days. The protective effects of induced-torpor were assessed via RNA sequencing and qPCR of mRNA extracted from the GIT. Pathway and network analysis were performed on the transcriptomic data to characterise the genomic signatures in radiation, torpor and torpor + radiation groups. Phenotypic analyses revealed that melatonin and reduced temperature successfully induced a torpor-like state in zebrafish as shown by decreased metabolism and activity levels. Genomic analyses indicated that low dose radiation caused DNA damage and oxidative stress triggering a stress response, including steroidal signalling and changes to metabolism, and cell cycle arrest. Torpor attenuated the stress response through an increase in pro-survival signals, reduced oxidative stress via the oxygen effect and detection and removal of misfolded proteins. This proof-of-concept model provides compelling initial evidence for utilizing an induced torpor-like state as a potential countermeasure for radiation exposure.

Functional Transcription Factor Target Networks Illuminate Control of Epithelial Remodelling.

Cell identity is governed by gene expression, regulated by transcription factor (TF) binding at cis-regulatory modules. Decoding the relationship between TF binding patterns and gene regulation is nontrivial, remaining a fundamental limitation in understanding cell decision-making. We developed the NetNC software to predict functionally active regulation of TF targets; demonstrated on nine datasets for the TFs Snail, Twist, and modENCODE Highly Occupied Target (HOT) regions. Snail and Twist are canonical drivers of epithelial to mesenchymal transition (EMT), a cell programme important in development, tumour progression and fibrosis. Predicted "neutral" (non-functional) TF binding always accounted for the majority (50% to 95%) of candidate target genes from statistically significant peaks and HOT regions had higher functional binding than most of the Snail and Twist datasets examined. Our results illuminated conserved gene networks that control epithelial plasticity in development and disease. We identified new gene functions and network modules including crosstalk with notch signalling and regulation of chromatin organisation, evidencing networks that reshape Waddington's epigenetic landscape during epithelial remodelling. Expression of orthologous functional TF targets discriminated breast cancer molecular subtypes and predicted novel tumour biology, with implications for precision medicine. Predicted invasion roles were validated using a tractable cell model, supporting our approach.

Overcoming intratumoural heterogeneity for reproducible molecular risk stratification: a case study in advanced kidney cancer.

BACKGROUND: Metastatic clear cell renal cell cancer (mccRCC) portends a poor prognosis and urgently requires better clinical tools for prognostication as well as for prediction of response to treatment. Considerable investment in molecular risk stratification has sought to overcome the performance ceiling encountered by methods restricted to traditional clinical parameters. However, replication of results has proven challenging, and intratumoural heterogeneity (ITH) may confound attempts at tissue-based stratification. METHODS: We investigated the influence of confounding ITH on the performance of a novel molecular prognostic model, enabled by pathologist-guided multiregion sampling (n = 183) of geographically separated mccRCC cohorts from the SuMR trial (development, n = 22) and the SCOTRRCC study (validation, n = 22). Tumour protein levels quantified by reverse phase protein array (RPPA) were investigated alongside clinical variables. Regularised wrapper selection identified features for Cox multivariate analysis with overall survival as the primary endpoint. RESULTS: The optimal subset of variables in the final stratification model consisted of N-cadherin, EPCAM, Age, mTOR (NEAT). Risk groups from NEAT had a markedly different prognosis in the validation cohort (log-rank p = 7.62 × 10-7; hazard ratio (HR) 37.9, 95% confidence interval 4.1-353.8) and 2-year survival rates (accuracy = 82%, Matthews correlation coefficient = 0.62). Comparisons with established clinico-pathological scores suggest favourable performance for NEAT (Net reclassification improvement 7.1% vs International Metastatic Database Consortium score, 25.4% vs Memorial Sloan Kettering Cancer Center score). Limitations include the relatively small cohorts and associated wide confidence intervals on predictive performance. Our multiregion sampling approach enabled investigation of NEAT validation when limiting the number of samples analysed per tumour, which significantly degraded performance. Indeed, sample selection could change risk group assignment for 64% of patients, and prognostication with one sample per patient performed only slightly better than random expectation (median logHR = 0.109). Low grade tissue was associated with 3.5-fold greater variation in predicted risk than high grade (p = 0.044). CONCLUSIONS: This case study in mccRCC quantitatively demonstrates the critical importance of tumour sampling for the success of molecular biomarker studies research where ITH is a factor. The NEAT model shows promise for mccRCC prognostication and warrants follow-up in larger cohorts. Our work evidences actionable parameters to guide sample collection (tumour coverage, size, grade) to inform the development of reproducible molecular risk stratification methods.

Quantitative Shotgun Proteomics Unveils Candidate Novel Esophageal Adenocarcinoma (EAC)-specific Proteins.

Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC).Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC.This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches.

Adaptation services of floodplains and wetlands under transformational climate change.

Adaptation services are the ecosystem processes and services that benefit people by increasing their ability to adapt to change. Benefits may accrue from existing but newly used services where ecosystems persist or from novel services supplied following ecosystem transformation. Ecosystem properties that enable persistence or transformation are important adaptation services because they support future options. The adaptation services approach can be applied to decisions on trade-offs between currently valued services and benefits from maintaining future options. For example, ecosystem functions and services of floodplains depend on river flows. In those regions of the world where climate change projections are for hotter, drier conditions, floods will be less frequent and floodplains will either persist, though with modified structure and function, or transform to terrestrial (flood-independent) ecosystems. Many currently valued ecosystem services will reduce in supply or become unavailable, but new options are provided by adaptation services. We present a case study from the Murray-Darling Basin, Australia, for operationalizing the adaptation services concept for floodplains and wetlands. We found large changes in flow and flood regimes are likely under a scenario of +1.6°C by 2030, even with additional water restored to rivers under the proposed Murray-Darling Basin Plan. We predict major changes to floodplain ecosystems, including contraction of riparian forests and woodlands and expansion of terrestrial, drought-tolerant vegetation communities. Examples of adaptation services under this scenario include substitution of irrigated agriculture with dryland cropping and floodplain grazing; mitigation of damage from rarer, extreme floods; and increased tourism, recreational, and cultural values derived from fewer, smaller wetlands that can be maintained with environmental flows. Management for adaptation services will require decisions on where intervention can enable ecosystem persistence and where transformation is inevitable. New ways of managing water that include consideration of the increasing importance of adaptation services requires major changes to decision-making that better account for landscape heterogeneity and large-scale change rather than attempting to maintain ecosystems in fixed states.

Sunitinib Treatment Exacerbates Intratumoral Heterogeneity in Metastatic Renal Cancer.

PURPOSE: The aim of this study was to investigate the effect of VEGF-targeted therapy (sunitinib) on molecular intratumoral heterogeneity (ITH) in metastatic clear cell renal cancer (mccRCC). EXPERIMENTAL DESIGN: Multiple tumor samples (n = 187 samples) were taken from the primary renal tumors of patients with mccRCC who were sunitinib treated (n = 23, SuMR clinical trial) or untreated (n = 23, SCOTRRCC study). ITH of pathologic grade, DNA (aCGH), mRNA (Illumina Beadarray) and candidate proteins (reverse phase protein array) were evaluated using unsupervised and supervised analyses (driver mutations, hypoxia, and stromal-related genes). ITH was analyzed using intratumoral protein variance distributions and distribution of individual patient aCGH and gene-expression clustering. RESULTS: Tumor grade heterogeneity was greater in treated compared with untreated tumors (P = 0.002). In unsupervised analysis, sunitinib therapy was not associated with increased ITH in DNA or mRNA. However, there was an increase in ITH for the driver mutation gene signature (DNA and mRNA) as well as increasing variability of protein expression with treatment (P < 0.05). Despite this variability, significant chromosomal and transcript changes to key targets of sunitinib, such as VHL, PBRM1, and CAIX, occurred in the treated samples. CONCLUSIONS: These findings suggest that sunitinib treatment has significant effects on the expression and ITH of key tumor and treatment specific genes/proteins in mccRCC. The results, based on primary tumor analysis, do not support the hypothesis that resistant clones are selected and predominate following targeted therapy.

Carbonic anhydrase 9 expression increases with vascular endothelial growth factor-targeted therapy and is predictive of outcome in metastatic clear cell renal cancer.

BACKGROUND: There is a lack of biomarkers to predict outcome with targeted therapy in metastatic clear cell renal cancer (mccRCC). This may be because dynamic molecular changes occur with therapy. OBJECTIVE: To explore if dynamic, targeted-therapy-driven molecular changes correlate with mccRCC outcome. DESIGN, SETTING, AND PARTICIPANTS: Multiple frozen samples from primary tumours were taken from sunitinib-naïve (n=22) and sunitinib-treated mccRCC patients (n=23) for protein analysis. A cohort (n=86) of paired, untreated and sunitinib/pazopanib-treated mccRCC samples was used for validation. Array comparative genomic hybridisation (CGH) analysis and RNA interference (RNAi) was used to support the findings. INTERVENTION: Three cycles of sunitinib 50mg (4 wk on, 2 wk off). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Reverse phase protein arrays (training set) and immunofluorescence automated quantitative analysis (validation set) assessed protein expression. RESULTS AND LIMITATIONS: Differential expression between sunitinib-naïve and treated samples was seen in 30 of 55 proteins (p<0.05 for each). The proteins B-cell CLL/lymphoma 2 (BCL2), mutL homolog 1 (MLH1), carbonic anhydrase 9 (CA9), and mechanistic target of rapamycin (mTOR) (serine/threonine kinase) had both increased intratumoural variance and significant differential expression with therapy. The validation cohort confirmed increased CA9 expression with therapy. Multivariate analysis showed high CA9 expression after treatment was associated with longer survival (hazard ratio: 0.48; 95% confidence interval, 0.26-0.87; p=0.02). Array CGH profiles revealed sunitinib was associated with significant CA9 region loss. RNAi CA9 silencing in two cell lines inhibited the antiproliferative effects of sunitinib. Shortcomings of the study include selection of a specific protein for analysis, and the specific time points at which the treated tissue was analysed. CONCLUSIONS: CA9 levels increase with targeted therapy in mccRCC. Lower CA9 levels are associated with a poor prognosis and possible resistance, as indicated by the validation cohort. PATIENT SUMMARY: Drug treatment of advanced kidney cancer alters molecular markers of treatment resistance. Measuring carbonic anhydrase 9 levels may be helpful in determining which patients benefit from therapy.

The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster.

RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar(5G1) null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar(5G1) mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar(5G1) null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability.

TMA Navigator: Network inference, patient stratification and survival analysis with tissue microarray data.

Tissue microarrays (TMAs) allow multiplexed analysis of tissue samples and are frequently used to estimate biomarker protein expression in tumour biopsies. TMA Navigator (www.tmanavigator.org) is an open access web application for analysis of TMA data and related information, accommodating categorical, semi-continuous and continuous expression scores. Non-biological variation, or batch effects, can hinder data analysis and may be mitigated using the ComBat algorithm, which is incorporated with enhancements for automated application to TMA data. Unsupervised grouping of samples (patients) is provided according to Gaussian mixture modelling of marker scores, with cardinality selected by Bayesian information criterion regularization. Kaplan-Meier survival analysis is available, including comparison of groups identified by mixture modelling using the Mantel-Cox log-rank test. TMA Navigator also supports network inference approaches useful for TMA datasets, which often constitute comparatively few markers. Tissue and cell-type specific networks derived from TMA expression data offer insights into the molecular logic underlying pathophenotypes, towards more effective and personalized medicine. Output is interactive, and results may be exported for use with external programs. Private anonymous access is available, and user accounts may be generated for easier data management.

The use of reverse phase protein arrays (RPPA) to explore protein expression variation within individual renal cell cancers.

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant. In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma as well as other solid tumors. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis. Protein based analysis of RCC is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

Addressing statistical biases in nucleotide-derived protein databases for proteogenomic search strategies.

Proteogenomics has the potential to advance genome annotation through high quality peptide identifications derived from mass spectrometry experiments, which demonstrate a given gene or isoform is expressed and translated at the protein level. This can advance our understanding of genome function, discovering novel genes and gene structure that have not yet been identified or validated. Because of the high-throughput shotgun nature of most proteomics experiments, it is essential to carefully control for false positives and prevent any potential misannotation. A number of statistical procedures to deal with this are in wide use in proteomics, calculating false discovery rate (FDR) and posterior error probability (PEP) values for groups and individual peptide spectrum matches (PSMs). These methods control for multiple testing and exploit decoy databases to estimate statistical significance. Here, we show that database choice has a major effect on these confidence estimates leading to significant differences in the number of PSMs reported. We note that standard target:decoy approaches using six-frame translations of nucleotide sequences, such as assembled transcriptome data, apparently underestimate the confidence assigned to the PSMs. The source of this error stems from the inflated and unusual nature of the six-frame database, where for every target sequence there exists five "incorrect" targets that are unlikely to code for protein. The attendant FDR and PEP estimates lead to fewer accepted PSMs at fixed thresholds, and we show that this effect is a product of the database and statistical modeling and not the search engine. A variety of approaches to limit database size and remove noncoding target sequences are examined and discussed in terms of the altered statistical estimates generated and PSMs reported. These results are of importance to groups carrying out proteogenomics, aiming to maximize the validation and discovery of gene structure in sequenced genomes, while still controlling for false positives.