BACKGROUND

Prediction of 3-dimensional protein structures from amino acid sequences represents one of the most important problems in computational structural biology. The community-wide Critical Assessment of Structure Prediction (CASP) experiments have been designed to obtain an objective assessment of the state-of-the-art of the field, where I-TASSER was ranked as the best method in the server section of the recent 7th CASP experiment. Our laboratory has since then received numerous requests about the public availability of the I-TASSER algorithm and the usage of the I-TASSER predictions.

RESULTS

An on-line version of I-TASSER is developed at the KU Center for Bioinformatics which has generated protein structure predictions for thousands of modeling requests from more than 35 countries. A scoring function (C-score) based on the relative clustering structural density and the consensus significance score of multiple threading templates is introduced to estimate the accuracy of the I-TASSER predictions. A large-scale benchmark test demonstrates a strong correlation between the C-score and the TM-score (a structural similarity measurement with values in [0, 1]) of the first models with a correlation coefficient of 0.91. Using a C-score cutoff>> -1.5 for the models of correct topology, both false positive and false negative rates are below 0.1. Combining C-score and protein length, the accuracy of the I-TASSER models can be predicted with an average error of 0.08 for TM-score and 2 A for RMSD.

CONCLUSIONS

The I-TASSER server has been developed to generate automated full-length 3D protein structural predictions where the benchmarked scoring system helps users to obtain quantitative assessments of the I-TASSER models. The output of the I-TASSER server for each query includes up to five full-length models, the confidence score, the estimated TM-score and RMSD, and the standard deviation of the estimations. The I-TASSER server is freely available to the academic community at http://zhang.bioinformatics.ku.edu/I-TASSER.

It is proposed that most neoplasms arise from a single cell of origin, and tumor progression results from acquired genetic variability within the original clone allowing sequential selection of more aggressive sublines. Tumor cell populations are apparently more genetically unstable than normal cells, perhaps from activation of specific gene loci in the neoplasm, continued presence of carcinogen, or even nutritional deficiencies within the tumor. The acquired genetic insta0ility and associated selection process, most readily recognized cytogenetically, results in advanced human malignancies being highly individual karyotypically and biologically. Hence, each patient's cancer may require individual specific therapy, and even this may be thwarted by emergence of a genetically variant subline resistant to the treatment. More research should be directed toward understanding and controlling the evolutionary process in tumors before it reaches the late stage usually seen in clinical cancer.

This note summarizes developments of the genepop software since its first description in 1995, and in particular those new to version 4.0: an extended input format, several estimators of neighbourhood size under isolation by distance, new estimators and confidence intervals for null allele frequency, and less important extensions to previous options. genepop now runs under Linux as well as under Windows, and can be entirely controlled by batch calls.

Accurate and automated methods for measuring the thickness of human cerebral cortex could provide powerful tools for diagnosing and studying a variety of neurodegenerative and psychiatric disorders. Manual methods for estimating cortical thickness from neuroimaging data are labor intensive, requiring several days of effort by a trained anatomist. Furthermore, the highly folded nature of the cortex is problematic for manual techniques, frequently resulting in measurement errors in regions in which the cortical surface is not perpendicular to any of the cardinal axes. As a consequence, it has been impractical to obtain accurate thickness estimates for the entire cortex in individual subjects, or group statistics for patient or control populations. Here, we present an automated method for accurately measuring the thickness of the cerebral cortex across the entire brain and for generating cross-subject statistics in a coordinate system based on cortical anatomy. The intersubject standard deviation of the thickness measures is shown to be less than 0.5 mm, implying the ability to detect focal atrophy in small populations or even individual subjects. The reliability and accuracy of this new method are assessed by within-subject test-retest studies, as well as by comparison of cross-subject regional thickness measures with published values.

A method for the screening of antioxidant activity is reported as a decolorization assay applicable to both lipophilic and hydrophilic antioxidants, including flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is reduced in the presence of such hydrogen-donating antioxidants. The influences of both the concentration of antioxidant and duration of reaction on the inhibition of the radical cation absorption are taken into account when determining the antioxidant activity. This assay clearly improves the original TEAC assay (the ferryl myoglobin/ABTS assay) for the determination of antioxidant activity in a number of ways. First, the chemistry involves the direct generation of the ABTS radical monocation with no involvement of an intermediary radical. Second, it is a decolorization assay; thus the radical cation is pre-formed prior to addition of antioxidant test systems, rather than the generation of the radical taking place continually in the presence of the antioxidant. Hence the results obtained with the improved system may not always be directly comparable with those obtained using the original TEAC assay. Third, it is applicable to both aqueous and lipophilic systems.

Synthesis of DNA at chromosome ends by telomerase may be necessary for indefinite proliferation of human cells. A highly sensitive assay for measuring telomerase activity was developed. In cultured cells representing 18 different human tissues, 98 of 100 immortal and none of 22 mortal populations were positive for telomerase. Similarly, 90 of 101 biopsies representing 12 human tumor types and none of 50 normal somatic tissues were positive. Normal ovaries and testes were positive, but benign tumors such as fibroids were negative. Thus, telomerase appears to be stringently repressed in normal human somatic tissues but reactivated in cancer, where immortal cells are likely required to maintain tumor growth.

An algorithm is presented for the multiple alignment of sequences, either proteins or nucleic acids, that is both accurate and easy to use on microcomputers. The approach is based on the conventional dynamic-programming method of pairwise alignment. Initially, a hierarchical clustering of the sequences is performed using the matrix of the pairwise alignment scores. The closest sequences are aligned creating groups of aligned sequences. Then close groups are aligned until all sequences are aligned in one group. The pairwise alignments included in the multiple alignment form a new matrix that is used to produce a hierarchical clustering. If it is different from the first one, iteration of the process can be performed. The method is illustrated by an example: a global alignment of 39 sequences of cytochrome c.

Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.

RNA is not only a messenger operating between DNA and protein. Transcription of essentially the entire eukaryotic genome generates a myriad of non-protein-coding RNA species that show complex overlapping patterns of expression and regulation. Although long noncoding RNAs (lncRNAs) are among the least well-understood of these transcript species, they cannot all be dismissed as merely transcriptional "noise." Here, we review the evolution of lncRNAs and their roles in transcriptional regulation, epigenetic gene regulation, and disease.

Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.

De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.

Agreement between two methods of clinical measurement can be quantified using the differences between observations made using the two methods on the same subjects. The 95% limits of agreement, estimated by mean difference +/- 1.96 standard deviation of the differences, provide an interval within which 95% of differences between measurements by the two methods are expected to lie. We describe how graphical methods can be used to investigate the assumptions of the method and we also give confidence intervals. We extend the basic approach to data where there is a relationship between difference and magnitude, both with a simple logarithmic transformation approach and a new, more general, regression approach. We discuss the importance of the repeatability of each method separately and compare an estimate of this to the limits of agreement. We extend the limits of agreement approach to data with repeated measurements, proposing new estimates for equal numbers of replicates by each method on each subject, for unequal numbers of replicates, and for replicated data collected in pairs, where the underlying value of the quantity being measured is changing. Finally, we describe a nonparametric approach to comparing methods.

Oncogenic ras can transform most immortal rodent cells to a tumorigenic state. However, transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors such as p53 or p16. Here we show that expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells, and E1A achieves a similar effect in human cells. These observations suggest that the onset of cellular senescence does not simply reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis.

Epithelial-mesenchymal transition is an indispensable mechanism during morphogenesis, as without mesenchymal cells, tissues and organs will never be formed. However, epithelial-cell plasticity, coupled to the transient or permanent formation of mesenchyme, goes far beyond the problem of cell-lineage segregation. Understanding how mesenchymal cells arise from an epithelial default status will also have a strong impact in unravelling the mechanisms that control fibrosis and cancer progression.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.

To pursue a systematic approach to the discovery of functional connections among diseases, genetic perturbation, and drug action, we have created the first installment of a reference collection of gene-expression profiles from cultured human cells treated with bioactive small molecules, together with pattern-matching software to mine these data. We demonstrate that this "Connectivity Map" resource can be used to find connections among small molecules sharing a mechanism of action, chemicals and physiological processes, and diseases and drugs. These results indicate the feasibility of the approach and suggest the value of a large-scale community Connectivity Map project.

The anterior insular cortex (AIC) is implicated in a wide range of conditions and behaviours, from bowel distension and orgasm, to cigarette craving and maternal love, to decision making and sudden insight. Its function in the re-representation of interoception offers one possible basis for its involvement in all subjective feelings. New findings suggest a fundamental role for the AIC (and the von Economo neurons it contains) in awareness, and thus it needs to be considered as a potential neural correlate of consciousness.

Colon carcinoma is the second most common cause of death from cancer. The isolation and characterization of tumorigenic colon cancer cells may help to devise novel diagnostic and therapeutic procedures. Although there is increasing evidence that a rare population of undifferentiated cells is responsible for tumour formation and maintenance, this has not been explored for colorectal cancer. Here, we show that tumorigenic cells in colon cancer are included in the high-density CD133+ population, which accounts for about 2.5% of the tumour cells. Subcutaneous injection of colon cancer CD133+ cells readily reproduced the original tumour in immunodeficient mice, whereas CD133- cells did not form tumours. Such tumours were serially transplanted for several generations, in each of which we observed progressively faster tumour growth without significant phenotypic alterations. Unlike CD133- cells, CD133+ colon cancer cells grew exponentially for more than one year in vitro as undifferentiated tumour spheres in serum-free medium, maintaining the ability to engraft and reproduce the same morphological and antigenic pattern of the original tumour. We conclude that colorectal cancer is created and propagated by a small number of undifferentiated tumorigenic CD133+ cells, which should therefore be the target of future therapies.

Improvement in the clinical outcome of lung cancer is likely to be achieved by identification of the molecular events that underlie its pathogenesis. Here we show that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in non-small-cell lung cancer (NSCLC) cells. Mouse 3T3 fibroblasts forced to express this human fusion tyrosine kinase generated transformed foci in culture and subcutaneous tumours in nude mice. The EML4-ALK fusion transcript was detected in 6.7% (5 out of 75) of NSCLC patients examined; these individuals were distinct from those harbouring mutations in the epidermal growth factor receptor gene. Our data demonstrate that a subset of NSCLC patients may express a transforming fusion kinase that is a promising candidate for a therapeutic target as well as for a diagnostic molecular marker in NSCLC.

To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.

Each year, the American Cancer Society estimates the number of new cancer cases and deaths expected in the United States in the current year and compiles the most recent data on cancer incidence, mortality, and survival based on incidence data from the National Cancer Institute and mortality data from the National Center for Health Statistics. Incidence and death rates are age-standardized to the 2000 US standard million population. A total of 1,399,790 new cancer cases and 564,830 deaths from cancer are expected in the United States in 2006. When deaths are aggregated by age, cancer has surpassed heart disease as the leading cause of death for those younger than age 85 since 1999. Delay-adjusted cancer incidence rates stabilized in men from 1995 through 2002, but continued to increase by 0.3% per year from 1987 through 2002 in women. Between 2002 and 2003, the actual number of recorded cancer deaths decreased by 778 in men, but increased by 409 in women, resulting in a net decrease of 369, the first decrease in the total number of cancer deaths since national mortality record keeping was instituted in 1930. The death rate from all cancers combined has decreased by 1.5% per year since 1993 among men and by 0.8% per year since 1992 among women. The mortality rate has also continued to decrease for the three most common cancer sites in men (lung and bronchus, colon and rectum, and prostate) and for breast and colon and rectum cancers in women. Lung cancer mortality among women continues to increase slightly. In analyses by race and ethnicity, African American men and women have 40% and 18% higher death rates from all cancers combined than White men and women, respectively. Cancer incidence and death rates are lower in other racial and ethnic groups than in Whites and African Americans for all sites combined and for the four major cancer sites. However, these groups generally have higher rates for stomach, liver, and cervical cancers than Whites. Furthermore, minority populations are more likely to be diagnosed with advanced stage disease than are Whites. Progress in reducing the burden of suffering and death from cancer can be accelerated by applying existing cancer control knowledge across all segments of the population.

DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.

Schizophrenia is a highly heritable disorder. Genetic risk is conferred by a large number of alleles, including common alleles of small effect that might be detected by genome-wide association studies. Here we report a multi-stage schizophrenia genome-wide association study of up to 36,989 cases and 113,075 controls. We identify 128 independent associations spanning 108 conservatively defined loci that meet genome-wide significance, 83 of which have not been previously reported. Associations were enriched among genes expressed in brain, providing biological plausibility for the findings. Many findings have the potential to provide entirely new insights into aetiology, but associations at DRD2 and several genes involved in glutamatergic neurotransmission highlight molecules of known and potential therapeutic relevance to schizophrenia, and are consistent with leading pathophysiological hypotheses. Independent of genes expressed in brain, associations were enriched among genes expressed in tissues that have important roles in immunity, providing support for the speculated link between the immune system and schizophrenia.

The elucidation of the human genome sequence has made it possible to identify genetic alterations in cancers in unprecedented detail. To begin a systematic analysis of such alterations, we determined the sequence of well-annotated human protein-coding genes in two common tumor types. Analysis of 13,023 genes in 11 breast and 11 colorectal cancers revealed that individual tumors accumulate an average of approximately 90 mutant genes but that only a subset of these contribute to the neoplastic process. Using stringent criteria to delineate this subset, we identified 189 genes (average of 11 per tumor) that were mutated at significant frequency. The vast majority of these genes were not known to be genetically altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion. These data define the genetic landscape of two human cancer types, provide new targets for diagnostic and therapeutic intervention, and open fertile avenues for basic research in tumor biology.