A new immuno-TRAP technique overcomes limitations of spatial resolution and selection bias to identify gene locus associations with a nuclear subcompartment such as promyelocytic leukemia nuclear bodies.
Important insights into nuclear function would arise if gene loci physically interacting with particular subnuclear domains could be readily identified. Immunofluorescence microscopy combined with fluorescence in situ hybridization (immuno-FISH), the method that would typically be used in such a study, is limited by spatial resolution and requires prior assumptions for selecting genes to probe. Our new technique, immuno-TRAP, overcomes these limitations. Using promyelocytic leukemia nuclear bodies (PML NBs) as a model, we used immuno-TRAP to determine if specific genes localize within molecular dimensions with these bodies. Although we confirmed a TP53 gene–PML NB association, immuno-TRAP allowed us to uncover novel locus-PML NB associations, including the ABCA7 and TFF1 loci and, most surprisingly, the PML locus itself. These associations were cell type specific and reflected the cell’s physiological state. Combined with microarrays or deep sequencing, immuno-TRAP provides powerful opportunities for identifying gene locus associations with potentially any nuclear subcompartment.
Background and Aim
Liver cirrhosis is associated with decreased hepatic cytochrome P4503A (CYP3A) activity but the pathogenesis of this phenomenon is not well elucidated. In this study, we examined if certain microRNAs (miRNA) are associated with decreased hepatic CYP3A activity in cirrhosis.
Hepatic CYP3A activity and miRNA microarray expression profiles were measured in cirrhotic (n=28) and normal (n=12) liver tissue. Hepatic CYP3A activity was measured via midazolam hydroxylation in human liver microsomes. Additionally, hepatic CYP3A4 protein concentration and the expression of CYP3A4 mRNA were measured. Analyses were conducted to identify miRNAs which were differentially expressed between two groups but also were significantly associated with lower hepatic CYP3A activity.
Hepatic CYP3A activity in cirrhotic livers was 1.7-fold lower than in the normal livers (0.28 ± 0.06 vs. 0.47 ± 0.07mL* min-1*mg protein-1 (mean ± SEM), P=0.02). Six microRNAs (miR-155, miR-454, miR-582-5p, let-7f-1*, miR-181d, and miR-500) had >1.2-fold increase in cirrhotic livers and also had significant negative correlation with hepatic CYP3A activity (range of r = -0.44 to -0.52, P <0.05). Notably, miR-155, a known regulator of liver inflammation, had the highest fold increase in cirrhotic livers (2.2-fold, P=4.16E-08) and significantly correlated with hepatic CYP3A activity (r=-0.50, P=0.017). The relative expression (2-ΔΔCt mean ± SEM) of hepatic CYP3A4 mRNA was significantly higher in cirrhotic livers (21.76 ± 2.65 vs. 5.91 ± 1.29, P=2.04E-07) but their levels did not significantly correlate with hepatic CYP3A activity (r=-0.43, P=0.08).
The strong association between certain miRNAs, notably miR-155, and lower hepatic CYP3A activity suggest that altered miRNA expression may regulate hepatic CYP3A activity.
Automated annotation of protein function is challenging. As the number of sequenced genomes rapidly grows, the overwhelming majority of protein products can only be annotated computationally. If computational predictions are to be relied upon, it is crucial that the accuracy of these methods be high. Here we report the results from the first large-scale community-based Critical Assessment of protein Function Annotation (CAFA) experiment. Fifty-four methods representing the state-of-the-art for protein function prediction were evaluated on a target set of 866 proteins from eleven organisms. Two findings stand out: (i) today’s best protein function prediction algorithms significantly outperformed widely-used first-generation methods, with large gains on all types of targets; and (ii) although the top methods perform well enough to guide experiments, there is significant need for improvement of currently available tools.
atients undergoing resections for suspicious pulmonary lesions have a 9-55% benign rate. Validated prediction models exist to estimate the probability of malignancy in a general population and current practice guidelines recommend their use. We evaluated these models in a surgical population to determine the accuracy of existing models to predict benign or malignant disease.
We conducted a retrospective review of our thoracic surgery quality improvement database (2005-2008) to identify patients who underwent resection of a pulmonary lesion. Patients were stratified into subgroups based on age, smoking status and fluorodeoxyglucose positron emission tomography (PET) results. The probability of malignancy was calculated for each patient using the Mayo and SPN prediction models. Receiver operating characteristic (ROC) and calibration curves were used to measure model performance.
89 patients met selection criteria; 73% were malignant. Patients with preoperative PET scans were divided into 4 subgroups based on age, smoking history and nodule PET avidity. Older smokers with PET-avid lesions had a 90% malignancy rate. Patients with PET- non-avid lesions, or PET-avid lesions with age<50 years or never smokers of any age had a 62% malignancy rate. The area under the ROC curve for the Mayo and SPN models was 0.79 and 0.80, respectively; however, the models were poorly calibrated (p<0.001).
Despite improvements in diagnostic and imaging techniques, current general population models do not accurately predict lung cancer among patients ref erred for surgical evaluation. Prediction models with greater accuracy are needed to identify patients with benign disease to reduce non-therapeutic resections.
Lung Cancer; Lung Cancer; diagnosis; cancer staging; Positron Emission Tomography (PET)
Video-assisted thoracic surgery (VATS) lobectomy has become the standard of care for early stage lung cancer throughout the world. Teaching this complex procedure requires adequate case volume, adequate instrumentation, a committed operating room team and baseline experience with open lobectomy. We outline what key maneuvers and steps are required to teach and learn VATS lobectomy. This is most easily performed as part of a thoracic surgery training program, but with adequate commitment and proctoring, there is no reason experienced open surgeons cannot become proficient VATS surgeons. We provide videos showing the key portions of a subcarinal lymph node dissection, posterior hilar dissection of the right upper lobe, fissureless right middle lobectomy, and fissureless left lower lobectomy. These videos highlight what we feel are important principals in VATS lobectomy, i.e., N2 and N1 lymph node dissection, fissureless techniques, and progressive responsibility of the learner. Current literature in simulation of VATS lobectomy is also outlined as this will be the future of teaching in VATS lobectomy.
video-assisted thoracic surgery (VATS) lobectomy; teaching; simulation
The epithelial-to-mesenchymal transition (EMT) is a de-differentiation process that has been implicated in metastasis and the generation of cancer initiating cells (CICs) in solid tumors. To examine EMT in non-small cell lung cancer (NSCLC), we utilized a three dimensional (3D) cell culture system in which cells were co-stimulated with tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGFβ). NSCLC spheroid cultures display elevated expression of EMT master-switch transcription factors, TWIST1, SNAI1/Snail1, SNAI2/Slug and ZEB2/Sip1, and are highly invasive. Mesenchymal NSCLC cultures show CIC characteristics, displaying elevated expression of transcription factors KLF4, SOX2, POU5F1/Oct4, MYCN, and KIT. As a result, these putative CIC display a cancer “stem-like” phenotype by forming lung metastases under limiting cell dilution. The pleiotropic transcription factor, NF-κB, has been implicated in EMT and metastasis. Thus, we set out to develop a NSCLC model to further characterize the role of NF-κB activation in the development of CICs. Here, we demonstrate that induction of EMT in 3D cultures results in constitutive NF-κB activity. Furthermore, inhibition of NF-κB resulted in the loss of TWIST1, SNAI2, and ZEB2 induction, and a failure of cells to invade and metastasize. Our work indicates that NF-κB is required for NSCLC metastasis, in part, by transcriptionally upregulating master-switch transcription factors required for EMT.
Conodonts have been considered the earliest skeletonizing vertebrates and their mineralized feeding apparatus interpreted as having performed a tooth function. However, the absence of jaws in conodonts and the small size of their oropharyngeal musculature limits the force available for fracturing food items, presenting a challenge to this interpretation. We address this issue quantitatively using engineering approaches previously applied to mammalian dentitions. We show that the morphology of conodont food-processing elements was adapted to overcome size limitations through developing dental tools of unparalleled sharpness that maximize applied pressure. Combined with observations of wear, we also show how this morphology was employed, demonstrating how Wurmiella excavata used rotational kinematics similar to other conodonts, suggesting that this occlusal style is typical for the clade. Our work places conodont elements within a broader dental framework, providing a phylogenetically independent system for examining convergence and scaling in dental tools.
conodont; tooth; dental tools; finite-element analysis; Wurmiella excavata
The expansion of repressive epigenetic marks has been implicated in heterochromatin formation during embryonic development, but the general applicability of this mechanism is unclear. Here we show that nuclear rearrangement of repressive histone marks H3K9me3 and H3K27me3 into nonoverlapping structural layers characterizes senescence-associated heterochromatic foci (SAHF) formation in human fibroblasts. However, the global landscape of these repressive marks remains unchanged upon SAHF formation, suggesting that in somatic cells, heterochromatin can be formed through the spatial repositioning of pre-existing repressively marked histones. This model is reinforced by the correlation of presenescent replication timing with both the subsequent layered structure of SAHFs and the global landscape of the repressive marks, allowing us to integrate microscopic and genomic information. Furthermore, modulation of SAHF structure does not affect the occupancy of these repressive marks, nor vice versa. These experiments reveal that high-order heterochromatin formation and epigenetic remodeling of the genome can be discrete events.
The organisation of the large volume of mammalian genomic DNA within cell nuclei requires mechanisms to regulate chromatin compaction involving the reversible formation of higher order structures. The compaction state of chromatin varies between interphase and mitosis and is also subject to rapid and reversible change upon ATP depletion/repletion. In this study we have investigated mechanisms that may be involved in promoting the hyper-condensation of chromatin when ATP levels are depleted by treating cells with sodium azide and 2-deoxyglucose. Chromatin conformation was analysed in both live and permeabilised HeLa cells using FLIM-FRET, high resolution fluorescence microscopy and by electron spectroscopic imaging microscopy. We show that chromatin compaction following ATP depletion is not caused by loss of transcription activity and that it can occur at a similar level in both interphase and mitotic cells. Analysis of both live and permeabilised HeLa cells shows that chromatin conformation within nuclei is strongly influenced by the levels of divalent cations, including calcium and magnesium. While ATP depletion results in an increase in the level of unbound calcium, chromatin condensation still occurs even in the presence of a calcium chelator. Chromatin compaction is shown to be strongly affected by small changes in the levels of polyamines, including spermine and spermidine. The data are consistent with a model in which the increased intracellular pool of polyamines and divalent cations, resulting from depletion of ATP, bind to DNA and contribute to the large scale hyper-compaction of chromatin by a charge neutralisation mechanism.
The autoimmune liver disease primary biliary cirrhosis (PBC) is associated with life-altering fatigue in ∼50% of patients. Previous work suggests that fatigued PBC subjects have evidence of autonomic dysfunction and may be at a higher risk of sudden cardiac death. The manifestation of this risk is not clear. This pilot study investigated whether alterations in cardiac torsion and strain could be detected in fatigued or nonfatigued early-stage PBC patients. We performed cardiac tissue tagging and anatomical cine-imaging in 13 early-stage PBC patients (including 7 with significant fatigue) and 10 control subjects to calculate cardiac torsion and strain throughout systole and diastole. From the cardiac tagging, we calculated the torsion-to-shortening ratio (TSR), a measure of subepicardial torsion exerting mechanical advantage over subendocardial shortening. Autonomic function testing was performed to evaluate baroreceptor effective index on standing. TSR was markedly increased in the fatigued PBC patients (0.70 ± 0.13) compared with both controls (0.46 ± 0.11, P = 0.002) and nonfatigued PBC patients (0.44 ± 0.12, P = 0.003). Decreased baroreceptor effective index on standing strongly correlated with increased TSR within the whole PBC group (r = −0.71, P = 0.007). Fatigued PBC patients demonstrate a redistribution of myocardial strain characteristic of a reduced relative contribution to contraction from the subendocardium. This is analogous to the changes found in healthy aging for subjects ∼16 yr older than the fatigued PBC patients. Hence the hearts of fatigued PBC patients may be subject to processes of accelerated aging.
torsion; strain; magnetic resonance imaging; liver disease; autoimmune
The emergence of high-throughput, next-generation sequencing technologies has dramatically altered the way we assess genomes in population genetics and in cancer genomics. Currently, there are four commonly used whole-genome sequencing platforms on the market: Illumina’s HiSeq2000, Life Technologies’ SOLiD 4 and its completely redesigned 5500xl SOLiD, and Complete Genomics’ technology. A number of earlier studies have compared a subset of those sequencing platforms or compared those platforms with Sanger sequencing, which is prohibitively expensive for whole genome studies. Here we present a detailed comparison of the performance of all currently available whole genome sequencing platforms, especially regarding their ability to call SNVs and to evenly cover the genome and specific genomic regions. Unlike earlier studies, we base our comparison on four different samples, allowing us to assess the between-sample variation of the platforms. We find a pronounced GC bias in GC-rich regions for Life Technologies’ platforms, with Complete Genomics performing best here, while we see the least bias in GC-poor regions for HiSeq2000 and 5500xl. HiSeq2000 gives the most uniform coverage and displays the least sample-to-sample variation. In contrast, Complete Genomics exhibits by far the smallest fraction of bases not covered, while the SOLiD platforms reveal remarkable shortcomings, especially in covering CpG islands. When comparing the performance of the four platforms for calling SNPs, HiSeq2000 and Complete Genomics achieve the highest sensitivity, while the SOLiD platforms show the lowest false positive rate. Finally, we find that integrating sequencing data from different platforms offers the potential to combine the strengths of different technologies. In summary, our results detail the strengths and weaknesses of all four whole-genome sequencing platforms. It indicates application areas that call for a specific sequencing platform and disallow other platforms. This helps to identify the proper sequencing platform for whole genome studies with different application scopes.
Here, we present the new UCL Bioinformatics Group’s PSIPRED Protein Analysis Workbench. The Workbench unites all of our previously available analysis methods into a single web-based framework. The new web portal provides a greatly streamlined user interface with a number of new features to allow users to better explore their results. We offer a number of additional services to enable computationally scalable execution of our prediction methods; these include SOAP and XML-RPC web server access and new HADOOP packages. All software and services are available via the UCL Bioinformatics Group website at http://bioinf.cs.ucl.ac.uk/.
The outbreak of severe acute respiratory syndrome in 2002–2003 exacted considerable human and economic costs from countries involved. It also exposed major weaknesses in several of these countries in coping with an outbreak of a newly emerged infectious disease. In the 10 years since the outbreak, in addition to the increase in knowledge of the biology and epidemiology of this disease, a major lesson learned is the value of having a national public health institute that is prepared to control disease outbreaks and designed to coordinate a national response and assist localities in their responses.
severe acute respiratory syndrome; SARS; coronavirus; viruses; disease threats; public health; national public health institutes
Medulloblastoma is an aggressively-growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and displays tremendous biological and clinical heterogeneity1. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life.
Four tumour subgroups with distinct clinical, biological and genetic profiles are currently discriminated2,3. WNT tumours, displaying activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens4. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis2. Group 3 & 4 tumours are molecularly less well-characterised, and also present the greatest clinical challenges2,3,5. The full repertoire of genetic events driving this distinction, however, remains unclear.
Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs. Tetraploidy was identified as a frequent early event in Group 3 & 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA-sequencing confirmed these alterations, and revealed the expression of the first medulloblastoma fusion genes. Chromatin modifiers were frequently altered across all subgroups.
These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 & 4 patients.
To understand fully cell behaviour, biologists are making progress towards cataloguing the functional elements in the human genome and characterising their roles across a variety of tissues and conditions. Yet, functional information – either experimentally validated or computationally inferred by similarity – remains completely missing for approximately 30% of human proteins. FFPred was initially developed to bridge this gap by targeting sequences with distant or no homologues of known function and by exploiting clear patterns of intrinsic disorder associated with particular molecular activities and biological processes. Here, we present an updated and improved version, which builds on larger datasets of protein sequences and annotations, and uses updated component feature predictors as well as revised training procedures. FFPred 2.0 includes support vector regression models for the prediction of 442 Gene Ontology (GO) terms, which largely expand the coverage of the ontology and of the biological process category in particular. The GO term list mainly revolves around macromolecular interactions and their role in regulatory, signalling, developmental and metabolic processes. Benchmarking experiments on newly annotated proteins show that FFPred 2.0 provides more accurate functional assignments than its predecessor and the ProtFun server do; also, its assignments can complement information obtained using BLAST-based transfer of annotations, improving especially prediction in the biological process category. Furthermore, FFPred 2.0 can be used to annotate proteins belonging to several eukaryotic organisms with a limited decrease in prediction quality. We illustrate all these points through the use of both precision-recall plots and of the COGIC scores, which we recently proposed as an alternative numerical evaluation measure of function prediction accuracy.
The best current noninvasive surrogate for tumor biology is fluorodeoxyglucose positron emission tomography (FDG–PET). Both FDG–PET maximal standardized uptake values and selected tumor markers have been shown to correlate with stage, nodal disease, and survival in non–small cell lung cancer (NSCLC). However, there are limited data correlating FDG–PET with tumor markers. The purpose of this study was to determine the correlation of tumor marker expression with FDG–PET maximal standardized uptake values in NSCLC.
FDG–PET maximal standardized uptake values were calculated in patients with NSCLC (n = 149). No patient had induction chemoradiotherapy. Intraoperative NSCLC tissue was obtained and tissue microarrays were created. Immunohistochemical analysis was performed for 5 known NSCLC tumor markers (glucose transporter 1, p53, cyclin D1, epidermal growth factor receptor, and vascular endothelial growth factor). Each tumor marker was assessed independently by two pathologists using common grading criteria. Subgroup analysis based on histologic characteristics and regional nodal status was performed.
FDG–PET correlated with T classification (P<.0001), N stage (P = .002), and greatest tumor dimension (P<.0001). In addition, increasing maximal standardized uptake values correlated with increased expression of glucose transporter 1 (P<.0001) and p53 (P =.04) in adenocarcinoma. Epidermal growth factor receptor expression correlated with maximal standardized uptake values without predilection for histologic subtype (P = .004).
FDG–PET maximal standardized uptake values correlate with an increased expression of glucose transporter 1 and p53 in lung adenocarcinoma, but not squamous cell cancer. Future studies attempting to correlate FDG–PET with tumor biology will need to consider the effect of different tumor histologic types.
The University HealthSystem Consortium (UHC) mortality risk adjustment models are increasingly being used as benchmarks for quality assessment. But these administrative database models may include postoperative complications in their adjustments for preoperative risk. The purpose of this study was to compare the performance of the UHC with the Society of Thoracic Surgeons (STS) risk-adjusted mortality models for adult cardiac surgery and evaluate the contribution of postoperative complications on model performance.
We identified adult cardiac surgery patients with mortality risk estimates in both the UHC and Society of Thoracic Surgeons databases. We compared the predictive performance and calibration of estimates from both models. We then reestimated both models using only patients without any postoperative complications to determine the relative contribution of adjustments for postoperative events on model performance.
In the study population of 2,171 patients, the UHC model explained more variability (27% versus 13%, p < 0.001) and achieved better discrimination (C statistic = 0.88 versus 0.81, p < 0.001). But when applied in the population of patients without complications, the UHC model performance declined severely. The C statistic decreased from 0.88 to 0.49, a level of discrimination equivalent to random chance. The discrimination of the Society of Thoracic Surgeons model was unchanged (C statistic of 0.79 versus 0.81).
Although the UHC model demonstrated better performance in the total study population, this difference in performance reflects adjustments for conditions that are postoperative complications. The current UHC models should not be used for quality benchmarks.
We genotyped 2,861 cases from the UK PBC consortium and 8,514 UK population controls across 196,524 variants within 186 known autoimmune risk loci. We identified three loci newly associated with primary biliary cirrhosis (PBC) (with P<5×10−8), increasing the number of known susceptibility loci to 25. The most associated variant at 19p12 is a low-frequency non-synonymous SNP in TYK2, further implicating JAK/STAT and cytokine signalling in disease pathogenesis. A further five loci contained non-synonymous variants in high linkage disequilibrium (LD) (r2>0.8) with the most associated variant at the locus. We found multiple independent common, low-frequency and rare variant association signals at five loci. Of the 26 independent non-HLA signals tagged on Immunochip, 15 have SNPs in B-lymphoblastoid open-chromatin regions in high LD (r2>0.8) with the most associated variant. This study demonstrates how dense fine-mapping arrays coupled with functional genomic data can be utilized to identify candidate causal variants for functional follow-up.
Accurate protein function annotation is a severe bottleneck when utilizing the deluge of high-throughput, next generation sequencing data. Keeping database annotations up-to-date has become a major scientific challenge that requires the development of reliable automatic predictors of protein function. The CAFA experiment provided a unique opportunity to undertake comprehensive 'blind testing' of many diverse approaches for automated function prediction. We report on the methodology we used for this challenge and on the lessons we learnt.
Our method integrates into a single framework a wide variety of biological information sources, encompassing sequence, gene expression and protein-protein interaction data, as well as annotations in UniProt entries. The methodology transfers functional categories based on the results from complementary homology-based and feature-based analyses. We generated the final molecular function and biological process assignments by combining the initial predictions in a probabilistic manner, which takes into account the Gene Ontology hierarchical structure.
We propose a novel scoring function called COmbined Graph-Information Content similarity (COGIC) score for the comparison of predicted functional categories and benchmark data. We demonstrate that our integrative approach provides increased scope and accuracy over both the component methods and the naïve predictors. In line with previous studies, we find that molecular function predictions are more accurate than biological process assignments.
Overall, the results indicate that there is considerable room for improvement in the field. It still remains for the community to invest a great deal of effort to make automated function prediction a useful and routine component in the toolbox of life scientists. As already witnessed in other areas, community-wide blind testing experiments will be pivotal in establishing standards for the evaluation of prediction accuracy, in fostering advancements and new ideas, and ultimately in recording progress.
Recurrent mutations affecting the histone H3.3 residues Lys27 or indirectly Lys36 are frequent drivers of pediatric high-grade gliomas (over 30 % of HGGs). To identify additional driver mutations in HGGs, we investigated a cohort of 60 pediatric HGGs using whole-exome sequencing (WES) and compared them to 543 exomes from non-cancer control samples. We identified mutations in SETD2, a H3K36 trimethyltransferase, in 15 % of pediatric HGGs, a result that was genome-wide significant (FDR = 0.029). Most SETD2 alterations were truncating mutations. Sequencing the gene in this cohort and another validation cohort (123 gliomas from all ages and grades) showed SETD2 mutations to be specific to high-grade tumors affecting 15 % of pediatric HGGs (11/73) and 8 % of adult HGGs (5/65) while no SETD2 mutations were identified in low-grade diffuse gliomas (0/45). Furthermore, SETD2 mutations were mutually exclusive with H3F3A mutations in HGGs (P = 0.0492) while they partly overlapped with IDH1 mutations (4/14), and SETD2-mutant tumors were found exclusively in the cerebral hemispheres (P = 0.0055). SETD2 is the only H3K36 trimethyltransferase in humans, and SETD2-mutant tumors showed a substantial decrease in H3K36me3 levels (P < 0.001), indicating that the mutations are loss-of-function. These data suggest that loss-of-function SETD2 mutations occur in older children and young adults and are specific to HGG of the cerebral cortex, similar to the H3.3 G34R/V and IDH mutations. Taken together, our results suggest that mutations disrupting the histone code at H3K36, including H3.3 G34R/V, IDH1 and/or SETD2 mutations, are central to the genesis of hemispheric HGGs in older children and young adults.
Electronic supplementary material
The online version of this article (doi:10.1007/s00401-013-1095-8) contains supplementary material, which is available to authorized users.
High-grade glioma; H3K36 methylation; SETD2; Epigenetic; Pediatric; Young adult