Crowd-sourced benchmarking of single-sample tumor subclonal reconstruction.

Subclonal reconstruction algorithms use bulk DNA sequencing data to quantify parameters of tumor evolution, allowing an assessment of how cancers initiate, progress and respond to selective pressures. We launched the ICGC-TCGA (International Cancer Genome Consortium-The Cancer Genome Atlas) DREAM Somatic Mutation Calling Tumor Heterogeneity and Evolution Challenge to benchmark existing subclonal reconstruction algorithms. This 7-year community effort used cloud computing to benchmark 31 subclonal reconstruction algorithms on 51 simulated tumors. Algorithms were scored on seven independent tasks, leading to 12,061 total runs. Algorithm choice influenced performance substantially more than tumor features but purity-adjusted read depth, copy-number state and read mappability were associated with the performance of most algorithms on most tasks. No single algorithm was a top performer for all seven tasks and existing ensemble strategies were unable to outperform the best individual methods, highlighting a key research need. All containerized methods, evaluation code and datasets are available to support further assessment of the determinants of subclonal reconstruction accuracy and development of improved methods to understand tumor evolution.

Primary Serous Cystadenocarcinoma of the Spleen.

Tumours of the spleen are uncommon, and most are metastases from primaries in other organs. Primary splenic malignancies are subdivided into two main groups: lymphoid and non-lymphoid. Primary splenic cystadenocarcinomas are extremely rare, and only reports of the mucinous variant exist. We present the case of a female in her eighth decade of life who was found to have an incidental complex splenic mass with a cystic component, which showed an interval increase in size on serial imaging. After further investigation, including positron emission tomography (PET), endoscopic ultrasound (EUS), and laparoscopy, she successfully underwent distal pancreatectomy, splenectomy, and partial gastrectomy for a suspected locally invasive pancreatic malignancy. Histology and immunohistochemical analyses were consistent with the first recorded case of primary serous cystadenocarcinoma of the spleen in the literature.

A community challenge to evaluate RNA-seq, fusion detection, and isoform quantification methods for cancer discovery.

The accurate identification and quantitation of RNA isoforms present in the cancer transcriptome is key for analyses ranging from the inference of the impacts of somatic variants to pathway analysis to biomarker development and subtype discovery. The ICGC-TCGA DREAM Somatic Mutation Calling in RNA (SMC-RNA) challenge was a crowd-sourced effort to benchmark methods for RNA isoform quantification and fusion detection from bulk cancer RNA sequencing (RNA-seq) data. It concluded in 2018 with a comparison of 77 fusion detection entries and 65 isoform quantification entries on 51 synthetic tumors and 32 cell lines with spiked-in fusion constructs. We report the entries used to build this benchmark, the leaderboard results, and the experimental features associated with the accurate prediction of RNA species. This challenge required submissions to be in the form of containerized workflows, meaning each of the entries described is easily reusable through CWL and Docker containers at https://github.com/SMC-RNA-challenge. A record of this paper's transparent peer review process is included in the supplemental information.

A community effort to create standards for evaluating tumor subclonal reconstruction.

Tumor DNA sequencing data can be interpreted by computational methods that analyze genomic heterogeneity to infer evolutionary dynamics. A growing number of studies have used these approaches to link cancer evolution with clinical progression and response to therapy. Although the inference of tumor phylogenies is rapidly becoming standard practice in cancer genome analyses, standards for evaluating them are lacking. To address this need, we systematically assess methods for reconstructing tumor subclonality. First, we elucidate the main algorithmic problems in subclonal reconstruction and develop quantitative metrics for evaluating them. Then we simulate realistic tumor genomes that harbor all known clonal and subclonal mutation types and processes. Finally, we benchmark 580 tumor reconstructions, varying tumor read depth, tumor type and somatic variant detection. Our analysis provides a baseline for the establishment of gold-standard methods to analyze tumor heterogeneity.

Reproducible biomedical benchmarking in the cloud: lessons from crowd-sourced data challenges.

Challenges are achieving broad acceptance for addressing many biomedical questions and enabling tool assessment. But ensuring that the methods evaluated are reproducible and reusable is complicated by the diversity of software architectures, input and output file formats, and computing environments. To mitigate these problems, some challenges have leveraged new virtualization and compute methods, requiring participants to submit cloud-ready software packages. We review recent data challenges with innovative approaches to model reproducibility and data sharing, and outline key lessons for improving quantitative biomedical data analysis through crowd-sourced benchmarking challenges.

Long noncoding RNAs and sulforaphane: a target for chemoprevention and suppression of prostate cancer.

Long noncoding RNAs (lncRNAs) have emerged as important in cancer development and progression. The impact of diet on lncRNA expression is largely unknown. Sulforaphane (SFN), obtained from vegetables like broccoli, can prevent and suppress cancer formation. Here we tested the hypothesis that SFN attenuates the expression of cancer-associated lncRNAs. We analyzed whole-genome RNA-sequencing data of normal human prostate epithelial cells and prostate cancer cells treated with 15 µM SFN or dimethylsulfoxide. SFN significantly altered expression of ~100 lncRNAs in each cell type and normalized the expression of some lncRNAs that were differentially expressed in cancer cells. SFN-mediated alterations in lncRNA expression correlated with genes that regulate cell cycle, signal transduction and metabolism. LINC01116 was functionally investigated because it was overexpressed in several cancers, and was transcriptionally repressed after SFN treatment. Knockdown of LINC01116 with siRNA decreased proliferation of prostate cancer cells and significantly up-regulated several genes including GAPDH (regulates glycolysis), MAP1LC3B2 (autophagy) and H2AFY (chromatin structure). A four-fold decrease in the ability of the cancer cells to form colonies was found when the LINC01116 gene was disrupted through a CRISPR/CAS9 method, further supporting an oncogenic function for LINC01116 in PC-3 cells. We identified a novel isoform of LINC01116 and bioinformatically investigated the possibility that LINC01116 could interact with target genes via ssRNA:dsDNA triplexes. Our data reveal that chemicals from the diet can influence the expression of functionally important lncRNAs, and suggest a novel mechanism by which SFN may prevent and suppress prostate cancer.

Phloroglucinol functions as an intracellular and intercellular chemical messenger influencing gene expression in Pseudomonas protegens.

Bacteria can be both highly communicative and highly competitive in natural habitats and antibiotics are thought to play a role in both of these processes. The soil bacterium Pseudomonas protegens Pf-5 produces a spectrum of antibiotics, two of which, pyoluteorin and 2,4-diacetylphloroglucinol (DAPG), function in intracellular and intercellular communication, both as autoinducers of their own production. Here, we demonstrate that phloroglucinol, an intermediate in DAPG biosynthesis, can serve as an intercellular signal influencing the expression of pyoluteorin biosynthesis genes, the production of pyoluteorin, and inhibition of Pythium ultimum, a phytopathogenic oomycete sensitive to pyoluteorin. Through analysis of RNAseq data sets, we show that phloroglucinol had broad effects on the transcriptome of Pf-5, significantly altering the transcription of more than two hundred genes. The effects of nanomolar versus micromolar concentrations of phloroglucinol differed both quantitatively and qualitatively, influencing the expression of distinct sets of genes or having opposite effects on transcript abundance of certain genes. Therefore, our results support the concept of hormesis, a phenomenon associated with signalling molecules that elicit distinct responses at different concentrations. Phloroglucinol is the first example of an intermediate of antibiotic biosynthesis that functions as a chemical messenger influencing gene expression in P. protegens.

RNA-Seq analysis of resistant and susceptible potato varieties during the early stages of potato virus Y infection.

BACKGROUND: Potato virus Y (PVY) is one of the most important plant viruses affecting potato production. The interactions between potato and PVY are complex and the outcome of the interactions depends on the potato genotype, the PVY strain, and the environmental conditions. A potato cultivar can induce resistance to a specific PVY strain, yet be susceptible to another. How a single potato cultivar responds to PVY in both compatible and incompatible interactions is not clear. RESULTS: In this study, we used RNA-sequencing (RNA-Seq) to investigate and compare the transcriptional changes in leaves of potato upon inoculation with PVY. We used two potato varieties: Premier Russet, which is resistant to the PVY strain O (PVY(O)) but susceptible to the strain NTN (PVY(NTN)), and Russet Burbank, which is susceptible to all PVY strains that have been tested. Leaves were inoculated with PVY(O) or PVY(NTN), and samples were collected 4 and 10 h post inoculation (hpi). A larger number of differentially expressed (DE) genes were found in the compatible reactions compared to the incompatible reaction. For all treatments, the majority of DE genes were down-regulated at 4 hpi and up-regulated at 10 hpi. Gene Ontology enrichment analysis showed enrichment of the biological process GO term "Photosynthesis, light harvesting" specifically in PVY(O)-inoculated Premier Russet leaves, while the GO term "nucleosome assembly" was largely overrepresented in PVY(NTN)-inoculated Premier Russet leaves and PVY(O)-inoculated Russet Burbank leaves but not in PVY(O)-inoculated Premier Russet leaves. Fewer genes were DE over 4-fold in the incompatible reaction compared to the compatible reactions. Amongst these, five genes were DE only in PVY(O)-inoculated Premier Russet leaves, and all five were down-regulated. These genes are predicted to encode for a putative ABC transporter, a MYC2 transcription factor, a VQ-motif containing protein, a non-specific lipid-transfer protein, and a xyloglucan endotransglucosylase-hydroxylase. CONCLUSIONS: Our results show that the incompatible and compatible reactions in Premier Russet shared more similarities, in particular during the initial response, than the compatible reactions in the two different hosts. Our results identify potential key processes and genes that determine the fate of the reaction, compatible or incompatible, between PVY and its host.

Nitrobacter winogradskyi transcriptomic response to low and high ammonium concentrations.

Nitrobacter winogradskyi Nb-255 is a nitrite-oxidizing bacterium that can grow solely on nitrite (NO2(-)) as a source of energy and nitrogen. In most natural situations, NO2(-) oxidation is coupled closely to ammonium (NH4(+)) oxidation by bacteria and archaea and, conceptually, N. winogradskyi can save energy using NH4(+) to meet its N-biosynthetic requirements. Interestingly, NH4(+) delayed the growth of N. winogradskyi when at concentrations higher than 35 mM, but grew well at concentrations below 25 mM NH4(+) while adjusting the expression of 24% of its genes. Notable genes that changed in expression included those with roles in nitrogen and carbon assimilation. Contrary to expectations, higher expression of glutamate synthase (GOGAT), instead of glutamate dehydrogenase, was detected at higher NH4(+) concentration. Genes in assimilatory NO2(-) metabolism and the degradation of glycogen and biofilm/motility were downregulated when N. winogradskyi was grown in the presence of NH4(+). Nitrobacter winogradskyi grown in medium with 25 mM NH4(+) upregulated genes in post-translational modification, protein turnover, biogenesis and chaperons. The data suggest that N. winogradskyi physiology is modified in the presence of NH4(+) and is likely to be modified during coupled nitrification with NH3 oxidizers.

Interactions of Nitrosomonas europaea and Nitrobacter winogradskyi grown in co-culture.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH4 (+) resulted in a cell density (0.20-0.29 OD600) greater than the sum of the densities in single chemostat cultures, i.e., 0.09-0.14 OD600 for N. europaea with 60 mM NH4 (+)and 0.04-0.06 OD600 for N. winogradskyi with 60 mM NO2 (-). The NO2 (-)- and NH4 (+)-dependent O2 uptake rates, qRT-PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD600 (~80 %) and N. winogradskyi ~0.05 OD600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

Alternative Splicing in the Obligate Biotrophic Oomycete Pathogen Pseudoperonospora cubensis.

Pseudoperonospora cubensis is an obligate pathogen and causative agent of cucurbit downy mildew. To help advance our understanding of the pathogenicity of P. cubensis, we used RNA-Seq to improve the quality of its reference genome sequence. We also characterized the RNA-Seq dataset to inventory transcript isoforms and infer alternative splicing during different stages of its development. Almost half of the original gene annotations were improved and nearly 4,000 previously unannotated genes were identified. We also demonstrated that approximately 24% of the expressed genome and nearly 55% of the intron-containing genes from P. cubensis had evidence for alternative splicing. Our analyses revealed that intron retention is the predominant alternative splicing type in P. cubensis, with alternative 5'- and alternative 3'-splice sites occurring at lower frequencies. Representatives of the newly identified genes and predicted alternatively spliced transcripts were experimentally validated. The results presented herein highlight the utility of RNA-Seq for improving draft genome annotations and, through this approach, we demonstrate that alternative splicing occurs more frequently than previously predicted. In total, the current study provides evidence that alternative splicing plays a key role in transcriptome regulation and proteome diversification in plant-pathogenic oomycetes.

Transcriptome analysis reveals a dynamic and differential transcriptional response to sulforaphane in normal and prostate cancer cells and suggests a role for Sp1 in chemoprevention.

SCOPE: Epidemiological studies provide evidence that consumption of cruciferous vegetables, like broccoli, can reduce the risk of cancer development. Sulforaphane (SFN) is a phytochemical derived from cruciferous vegetables that induces anti-proliferative and pro-apoptotic responses in prostate cancer cells, but not in normal prostate cells. The mechanisms responsible for this cancer-specific cytotoxicity remain unclear. METHODS AND RESULTS: We utilized RNA sequencing and determined the transcriptomes of normal prostate epithelial cells, androgen-dependent prostate cancer cells, and androgen-independent prostate cancer cells treated with SFN. SFN treatment dynamically altered gene expression and resulted in distinct transcriptome profiles depending on prostate cell line. SFN also down-regulated the expression of genes that were up-regulated in prostate cancer cells. Network analysis of genes altered by SFN treatment revealed that the transcription factor Specificity protein 1 (Sp1) was present in an average of 90.5% of networks. Sp1 protein was significantly decreased by SFN treatment in prostate cancer cells and Sp1 may be an important mediator of SFN-induced changes in expression. CONCLUSION: Overall, the data show that SFN alters gene expression differentially in normal and cancer cells with key targets in chemopreventive processes, making it a promising dietary anti-cancer agent.

Effects of sulforaphane and 3,3'-diindolylmethane on genome-wide promoter methylation in normal prostate epithelial cells and prostate cancer cells.

Epigenetic changes, including aberrant DNA methylation, result in altered gene expression and play an important role in carcinogenesis. Phytochemicals such as sulforaphane (SFN) and 3,3'-diindolylmethane (DIM) are promising chemopreventive agents for the treatment of prostate cancer. Both have been shown to induce re-expression of genes, including tumor suppressor genes silenced in cancer cells, via modulation of epigenetic marks including DNA methylation. However, it remained unclear the effects SFN and DIM on DNA methylation at a genomic scale. The goal of this study was to determine the genome-wide effects of SFN and DIM on promoter methylation in normal prostate epithelial cells and prostate cancer cells. Both SFN and DIM treatment decreased DNA methyltransferase expression in normal prostate epithelial cells (PrEC), and androgen-dependent (LnCAP) and androgen-independent (PC3) prostate cancer cells. The effects of SFN and DIM on promoter methylation profiles in normal PrEC, LnCAP and PC3 prostate cancer cells were determined using methyl-DNA immunoprecipitation followed by genome-wide DNA methylation array. We showed widespread changes in promoter methylation patterns, including both increased and decreased methylation, in all three prostate cell lines in response to SFN or DIM treatments. In particular, SFN and DIM altered promoter methylation in distinct sets of genes in PrEC, LnCAP, and PC3 cells, but shared similar gene targets within a single cell line. We further showed that SFN and DIM reversed many of the cancer-associated methylation alterations, including aberrantly methylated genes that are dysregulated or are highly involved in cancer progression. Overall, our data suggested that both SFN and DIM are epigenetic modulators that have broad and complex effects on DNA methylation profiles in both normal and cancerous prostate epithelial cells. Results from our study may provide new insights into the epigenetic mechanisms by which SFN and DIM exert their cancer chemopreventive effects.

One-year audit of complaints made against a University Hospital Surgical Department.

BACKGROUND: There is relatively little in the medical literature relating to complaints about the healthcare process. The aim of this study was to report the frequency and content of patient complaints against a University Hospital Surgical Department. In particular, the study aimed to relate the number of complaints to the number of health-care episodes and to determine the frequency of patient safety incidents and subsequent medico-legal action. METHODS: Retrospective interrogation of a prospectively maintained Complaints Department database at a University Hospital for the calendar year 2009. RESULTS: Complaints relating to 360 aspects of the health-care journey in 113 patients were made. This translated into one complaint per 400 health-care episodes. Concerns about clinical care were cited in 31%, delays in the health-care process in 30%, communication issues in 19%, the institutional environment in 8% and poor discharge planning in 6%. Overall, 16 complaints (4%) were raised as patient safety incidents. Eighty-three per cent of complaints were addressed by a telephone conversation or a single letter response, 13% by a face-to-face meeting. Two per cent resulted in subsequent medico-legal action. CONCLUSIONS: Although perceived in a negative way by health-care professionals, only 1 in 400 health-care episodes resulted in a complaint. Only a small number related to patient safety incidents or resulted in medico-legal instructions. Attention should focus on developing effective strategies to improve patient satisfaction with all aspects of the patient journey.

Peatmoss (Sphagnum) diversification associated with Miocene Northern Hemisphere climatic cooling?

Global climate changes sometimes spark biological radiations that can feed back to effect significant ecological impacts. Northern Hemisphere peatlands dominated by living and dead peatmosses (Sphagnum) harbor almost 30% of the global soil carbon pool and have functioned as a net carbon sink throughout the Holocene, and probably since the late Tertiary. Before that time, northern latitudes were dominated by tropical and temperate plant groups and ecosystems. Phylogenetic analyses of mosses (phylum Bryophyta) based on nucleotide sequences from the plastid, mitochondrial, and nuclear genomes indicate that most species of Sphagnum are of recent origin (ca. <20 Ma). Sphagnum species are not only well-adapted to boreal peatlands, they create the conditions that promote development of peatlands. The recent radiation that gave rise to extant diversity of peatmosses is temporally associated with Miocene climatic cooling in the Northern Hemisphere. The evolution of Sphagnum has had profound influences on global biogeochemistry because of the unique biochemical, physiological, and morphological features of these plants, both while alive and after death.

Newly resolved relationships in an early land plant lineage: Bryophyta class Sphagnopsida (peat mosses).

PREMISE OF THE STUDY: The Sphagnopsida, an early-diverging lineage of mosses (phylum Bryophyta), are morphologically and ecologically unique and have profound impacts on global climate. The Sphagnopsida are currently classified in two genera, Sphagnum (peat mosses) with some 350-500 species and Ambuchanania with one species. An analysis of phylogenetic relationships among species and genera in the Sphagnopsida were conducted to resolve major lineages and relationships among species within the Sphagnopsida. • METHODS: Phylogenetic analyses of nucleotide sequences from the nuclear, plastid, and mitochondrial genomes (11 704 nucleotides total) were conducted and analyzed using maximum likelihood and Bayesian inference employing seven different substitution models of varying complexity. • KEY RESULTS: Phylogenetic analyses resolved three lineages within the Sphagnopsida: (1) Sphagnum sericeum, (2) S. inretortum plus Ambuchanania leucobryoides, and (3) all remaining species of Sphagnum. Sister group relationships among these three clades could not be resolved, but the phylogenetic results indicate that the highly divergent morphology of A. leucobryoides is derived within the Sphagnopsida rather than plesiomorphic. A new classification is proposed for class Sphagnopsida, with one order (Sphagnales), three families, and four genera. • CONCLUSIONS: The Sphagnopsida are an old lineage within the phylum Bryophyta, but the extant species of Sphagnum represent a relatively recent radiation. It is likely that additional species critical to understanding the evolution of peat mosses await discovery, especially in the southern hemisphere.