MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

The Biological Networks Gene Ontology tool (BiNGO) is an open-source Java tool to determine which Gene Ontology (GO) terms are significantly overrepresented in a set of genes. BiNGO can be used either on a list of genes, pasted as text, or interactively on subgraphs of biological networks visualized in Cytoscape. BiNGO maps the predominant functional themes of the tested gene set on the GO hierarchy, and takes advantage of Cytoscape's versatile visualization environment to produce an intuitive and customizable visual representation of the results.

For the purposes of classification, it should be specified whether osteoarthritis (OA) of the knee is of unknown origin (idiopathic, primary) or is related to a known medical condition or event (secondary). Clinical criteria for the classification of idiopathic OA of the knee were developed through a multicenter study group. Comparison diagnoses included rheumatoid arthritis and other painful conditions of the knee, exclusive of referred or para-articular pain. Variables from the medical history, physical examination, laboratory tests, and radiographs were used to develop sets of criteria that serve different investigative purposes. In contrast to prior criteria, these proposed criteria utilize classification trees, or algorithms.

Although there are many commercially available statistical software packages, only a few implement a competing risk analysis or a proportional hazards regression model with time-dependent covariates, which are necessary in studies on hematopoietic SCT. In addition, most packages are not clinician friendly, as they require that commands be written based on statistical languages. This report describes the statistical software 'EZR' (Easy R), which is based on R and R commander. EZR enables the application of statistical functions that are frequently used in clinical studies, such as survival analyses, including competing risk analyses and the use of time-dependent covariates, receiver operating characteristics analyses, meta-analyses, sample size calculation and so on, by point-and-click access. EZR is freely available on our website (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html) and runs on both Windows (Microsoft Corporation, USA) and Mac OS X (Apple, USA). This report provides instructions for the installation and operation of EZR.

A simple method for generating cDNA libraries from submicrogram quantities of mRNA is described. It combines classical first-strand synthesis with the novel RNase H-DNA polymerase I-mediated second-strand synthesis [Okayama, H., and Berg, P., Mol. Cell. Biol. 2 (1982) 161-170]. Neither the elaborate vector-primer system nor the classical hairpin loop cleavage by S1 nuclease are used. cDNA thus made can be tailed and cloned without further purification or sizing. Cloning efficiencies can be as high as 10(6) recombinants generated per microgram mRNA, a considerable improvement over earlier methods. Using the fully sequenced 1300 nucleotide-long bovine preproenkephalin mRNA, we have established by sequencing that the method yields faithful full-length transcripts. This procedure considerably simplifies the establishment of cDNA libraries and thus the cloning of low-abundance mRNAs.

The outbreak of a novel betacoronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development, we determined a 3.5 Å-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells--cancer stem cells--possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 x 10(4) unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.

A large number of tiny noncoding RNAs have been cloned and named microRNAs (miRs). Recently, we have reported that miR-15a and miR-16a, located at 13q14, are frequently deleted and/or down-regulated in patients with B cell chronic lymphocytic leukemia, a disorder characterized by increased survival. To further investigate the possible involvement of miRs in human cancers on a genome-wide basis, we have mapped 186 miRs and compared their location to the location of previous reported nonrandom genetic alterations. Here, we show that miR genes are frequently located at fragile sites, as well as in minimal regions of loss of heterozygosity, minimal regions of amplification (minimal amplicons), or common breakpoint regions. Overall, 98 of 186 (52.5%) of miR genes are in cancer-associated genomic regions or in fragile sites. Moreover, by Northern blotting, we have shown that several miRs located in deleted regions have low levels of expression in cancer samples. These data provide a catalog of miR genes that may have roles in cancer and argue that the full complement of miRs in a genome may be extensively involved in cancers.

Protocols of systematic reviews and meta-analyses allow for planning and documentation of review methods, act as a guard against arbitrary decision making during review conduct, enable readers to assess for the presence of selective reporting against completed reviews, and, when made publicly available, reduce duplication of efforts and potentially prompt collaboration. Evidence documenting the existence of selective reporting and excessive duplication of reviews on the same or similar topics is accumulating and many calls have been made in support of the documentation and public availability of review protocols. Several efforts have emerged in recent years to rectify these problems, including development of an international register for prospective reviews (PROSPERO) and launch of the first open access journal dedicated to the exclusive publication of systematic review products, including protocols (BioMed Central's Systematic Reviews). Furthering these efforts and building on the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines, an international group of experts has created a guideline to improve the transparency, accuracy, completeness, and frequency of documented systematic review and meta-analysis protocols--PRISMA-P (for protocols) 2015. The PRISMA-P checklist contains 17 items considered to be essential and minimum components of a systematic review or meta-analysis protocol.This PRISMA-P 2015 Explanation and Elaboration paper provides readers with a full understanding of and evidence about the necessity of each item as well as a model example from an existing published protocol. This paper should be read together with the PRISMA-P 2015 statement. Systematic review authors and assessors are strongly encouraged to make use of PRISMA-P when drafting and appraising review protocols.

Regulatory T cells (Tregs) play an indispensable role in maintaining immunological unresponsiveness to self-antigens and in suppressing excessive immune responses deleterious to the host. Tregs are produced in the thymus as a functionally mature subpopulation of T cells and can also be induced from naive T cells in the periphery. Recent research reveals the cellular and molecular basis of Treg development and function and implicates dysregulation of Tregs in immunological disease.

Rapid progress in deciphering the biological mechanism of Alzheimer's disease (AD) has arisen from the application of molecular and cell biology to this complex disorder of the limbic and association cortices. In turn, new insights into fundamental aspects of protein biology have resulted from research on the disease. This beneficial interplay between basic and applied cell biology is well illustrated by advances in understanding the genotype-to-phenotype relationships of familial Alzheimer's disease. All four genes definitively linked to inherited forms of the disease to date have been shown to increase the production and/or deposition of amyloid beta-protein in the brain. In particular, evidence that the presenilin proteins, mutations in which cause the most aggressive form of inherited AD, lead to altered intramembranous cleavage of the beta-amyloid precursor protein by the protease called gamma-secretase has spurred progress toward novel therapeutics. The finding that presenilin itself may be the long-sought gamma-secretase, coupled with the recent identification of beta-secretase, has provided discrete biochemical targets for drug screening and development. Alternate and novel strategies for inhibiting the early mechanism of the disease are also emerging. The progress reviewed here, coupled with better ability to diagnose the disease early, bode well for the successful development of therapeutic and preventative drugs for this major public health problem.

In phylogenetics, the unrooted model of phylogeny and the strict molecular clock model are two extremes of a continuum. Despite their dominance in phylogenetic inference, it is evident that both are biologically unrealistic and that the real evolutionary process lies between these two extremes. Fortunately, intermediate models employing relaxed molecular clocks have been described. These models open the gate to a new field of "relaxed phylogenetics." Here we introduce a new approach to performing relaxed phylogenetic analysis. We describe how it can be used to estimate phylogenies and divergence times in the face of uncertainty in evolutionary rates and calibration times. Our approach also provides a means for measuring the clocklikeness of datasets and comparing this measure between different genes and phylogenies. We find no significant rate autocorrelation among branches in three large datasets, suggesting that autocorrelated models are not necessarily suitable for these data. In addition, we place these datasets on the continuum of clocklikeness between a strict molecular clock and the alternative unrooted extreme. Finally, we present analyses of 102 bacterial, 106 yeast, 61 plant, 99 metazoan, and 500 primate alignments. From these we conclude that our method is phylogenetically more accurate and precise than the traditional unrooted model while adding the ability to infer a timescale to evolution.

Systematic reviews and meta-analyses are essential to summarize evidence relating to efficacy and safety of health care interventions accurately and reliably. The clarity and transparency of these reports, however, is not optimal. Poor reporting of systematic reviews diminishes their value to clinicians, policy makers, and other users. Since the development of the QUOROM (QUality Of Reporting Of Meta-analysis) Statement--a reporting guideline published in 1999--there have been several conceptual, methodological, and practical advances regarding the conduct and reporting of systematic reviews and meta-analyses. Also, reviews of published systematic reviews have found that key information about these studies is often poorly reported. Realizing these issues, an international group that included experienced authors and methodologists developed PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) as an evolution of the original QUOROM guideline for systematic reviews and meta-analyses of evaluations of health care interventions. The PRISMA Statement consists of a 27-item checklist and a four-phase flow diagram. The checklist includes items deemed essential for transparent reporting of a systematic review. In this Explanation and Elaboration document, we explain the meaning and rationale for each checklist item. For each item, we include an example of good reporting and, where possible, references to relevant empirical studies and methodological literature. The PRISMA Statement, this document, and the associated Web site (http://www.prisma-statement.org/) should be helpful resources to improve reporting of systematic reviews and meta-analyses.

In recent years, many new cortical areas have been identified in the macaque monkey. The number of identified connections between areas has increased even more dramatically. We report here on (1) a summary of the layout of cortical areas associated with vision and with other modalities, (2) a computerized database for storing and representing large amounts of information on connectivity patterns, and (3) the application of these data to the analysis of hierarchical organization of the cerebral cortex. Our analysis concentrates on the visual system, which includes 25 neocortical areas that are predominantly or exclusively visual in function, plus an additional 7 areas that we regard as visual-association areas on the basis of their extensive visual inputs. A total of 305 connections among these 32 visual and visual-association areas have been reported. This represents 31% of the possible number of pathways if each area were connected with all others. The actual degree of connectivity is likely to be closer to 40%. The great majority of pathways involve reciprocal connections between areas. There are also extensive connections with cortical areas outside the visual system proper, including the somatosensory cortex, as well as neocortical, transitional, and archicortical regions in the temporal and frontal lobes. In the somatosensory/motor system, there are 62 identified pathways linking 13 cortical areas, suggesting an overall connectivity of about 40%. Based on the laminar patterns of connections between areas, we propose a hierarchy of visual areas and of somatosensory/motor areas that is more comprehensive than those suggested in other recent studies. The current version of the visual hierarchy includes 10 levels of cortical processing. Altogether, it contains 14 levels if one includes the retina and lateral geniculate nucleus at the bottom as well as the entorhinal cortex and hippocampus at the top. Within this hierarchy, there are multiple, intertwined processing streams, which, at a low level, are related to the compartmental organization of areas V1 and V2 and, at a high level, are related to the distinction between processing centers in the temporal and parietal lobes. However, there are some pathways and relationships (about 10% of the total) whose descriptions do not fit cleanly into this hierarchical scheme for one reason or another. In most instances, though, it is unclear whether these represent genuine exceptions to a strict hierarchy rather than inaccuracies or uncertainities in the reported assignment.

Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.

We have analyzed 5,088 bacterial 16S rRNA gene sequences from the distal intestinal (cecal) microbiota of genetically obese ob/ob mice, lean ob/+ and wild-type siblings, and their ob/+ mothers, all fed the same polysaccharide-rich diet. Although the majority of mouse gut species are unique, the mouse and human microbiota(s) are similar at the division (superkingdom) level, with Firmicutes and Bacteroidetes dominating. Microbial-community composition is inherited from mothers. However, compared with lean mice and regardless of kinship, ob/ob animals have a 50% reduction in the abundance of Bacteroidetes and a proportional increase in Firmicutes. These changes, which are division-wide, indicate that, in this model, obesity affects the diversity of the gut microbiota and suggest that intentional manipulation of community structure may be useful for regulating energy balance in obese individuals. The sequences reported in this paper have been deposited in the GenBank database [accession nos. DQ 014552--DQ 015671 (mothers) and AY 989911--AY 993908 (offspring)].

A method has been developed whereby a very large number of colonies of Escherichia coli carrying different hybrid plasmids can be rapidly screened to determine which hybrid plasmids contain a specified DNA sequence or genes. The colonies to be screened are formed on nitrocellulose filters, and, after a reference set of these colonies has been prepared by replica plating, are lysed and their DNA is denatured and fixed to the filter in situ. The resulting DNA-prints of the colonies are then hybridized to a radioactive RNA that defines the sequence or gene of interest, and the result of this hybridization is assayed by autoradiography. Colonies whose DNA-prints exhibit hybridization can then be picked from the reference plate. We have used this method to isolate clones of ColE1 hybrid plasmids that contain Drosophila melanogaster genes for 18 and 28S rRNAs. In principle, the method can be used to isolate any gene whose base sequence is represented in an available RNA.

Anterior cingulate cortex (ACC) is a part of the brain's limbic system. Classically, this region has been related to affect, on the basis of lesion studies in humans and in animals. In the late 1980s, neuroimaging research indicated that ACC was active in many studies of cognition. The findings from EEG studies of a focal area of negativity in scalp electrodes following an error response led to the idea that ACC might be the brain's error detection and correction device. In this article, these various findings are reviewed in relation to the idea that ACC is a part of a circuit involved in a form of attention that serves to regulate both cognitive and emotional processing. Neuroimaging studies showing that separate areas of ACC are involved in cognition and emotion are discussed and related to results showing that the error negativity is influenced by affect and motivation. In addition, the development of the emotional and cognitive roles of ACC are discussed, and how the success of this regulation in controlling responses might be correlated with cingulate size. Finally, some theories are considered about how the different subdivisions of ACC might interact with other cortical structures as a part of the circuits involved in the regulation of mental and emotional activity.

BACKGROUND

There is no consensus definition of acute renal failure (ARF) in critically ill patients. More than 30 different definitions have been used in the literature, creating much confusion and making comparisons difficult. Similarly, strong debate exists on the validity and clinical relevance of animal models of ARF; on choices of fluid management and of end-points for trials of new interventions in this field; and on how information technology can be used to assist this process. Accordingly, we sought to review the available evidence, make recommendations and delineate key questions for future studies.

METHODS

We undertook a systematic review of the literature using Medline and PubMed searches. We determined a list of key questions and convened a 2-day consensus conference to develop summary statements via a series of alternating breakout and plenary sessions. In these sessions, we identified supporting evidence and generated recommendations and/or directions for future research.

RESULTS

We found sufficient consensus on 47 questions to allow the development of recommendations. Importantly, we were able to develop a consensus definition for ARF. In some cases it was also possible to issue useful consensus recommendations for future investigations. We present a summary of the findings. (Full versions of the six workgroups' findings are available on the internet at http://www.ADQI.net)

CONCLUSIONS

Despite limited data, broad areas of consensus exist for the physiological and clinical principles needed to guide the development of consensus recommendations for defining ARF, selection of animal models, methods of monitoring fluid therapy, choice of physiological and clinical end-points for trials, and the possible role of information technology.

Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology. A susceptibility locus for Crohn's disease has been mapped to chromosome 16. Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes. These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region. NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.

OBJECTIVE

Recently, an increasing number of systematic reviews have been published in which the measurement properties of health status questionnaires are compared. For a meaningful comparison, quality criteria for measurement properties are needed. Our aim was to develop quality criteria for design, methods, and outcomes of studies on the development and evaluation of health status questionnaires.

METHODS

Quality criteria for content validity, internal consistency, criterion validity, construct validity, reproducibility, longitudinal validity, responsiveness, floor and ceiling effects, and interpretability were derived from existing guidelines and consensus within our research group.

RESULTS

For each measurement property a criterion was defined for a positive, negative, or indeterminate rating, depending on the design, methods, and outcomes of the validation study.

CONCLUSIONS

Our criteria make a substantial contribution toward defining explicit quality criteria for measurement properties of health status questionnaires. Our criteria can be used in systematic reviews of health status questionnaires, to detect shortcomings and gaps in knowledge of measurement properties, and to design validation studies. The future challenge will be to refine and complete the criteria and to reach broad consensus, especially on quality criteria for good measurement properties.

BACKGROUND

Oncogenic fusion genes consisting of EML4 and anaplastic lymphoma kinase (ALK) are present in a subgroup of non-small-cell lung cancers, representing 2 to 7% of such tumors. We explored the therapeutic efficacy of inhibiting ALK in such tumors in an early-phase clinical trial of crizotinib (PF-02341066), an orally available small-molecule inhibitor of the ALK tyrosine kinase.

METHODS

After screening tumor samples from approximately 1500 patients with non-small-cell lung cancer for the presence of ALK rearrangements, we identified 82 patients with advanced ALK-positive disease who were eligible for the clinical trial. Most of the patients had received previous treatment. These patients were enrolled in an expanded cohort study instituted after phase 1 dose escalation had established a recommended crizotinib dose of 250 mg twice daily in 28-day cycles. Patients were assessed for adverse events and response to therapy.

RESULTS

Patients with ALK rearrangements tended to be younger than those without the rearrangements, and most of the patients had little or no exposure to tobacco and had adenocarcinomas. At a mean treatment duration of 6.4 months, the overall response rate was 57% (47 of 82 patients, with 46 confirmed partial responses and 1 confirmed complete response); 27 patients (33%) had stable disease. A total of 63 of 82 patients (77%) were continuing to receive crizotinib at the time of data cutoff, and the estimated probability of 6-month progression-free survival was 72%, with no median for the study reached. The drug resulted in grade 1 or 2 (mild) gastrointestinal side effects.

CONCLUSIONS

The inhibition of ALK in lung tumors with the ALK rearrangement resulted in tumor shrinkage or stable disease in most patients. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Control elements of the tetracycline-resistance operon encoded in Tn10 of Escherichia coli have been utilized to establish a highly efficient regulatory system in mammalian cells. By fusing the tet repressor with the activating domain of virion protein 16 of herpes simplex virus, a tetracycline-controlled transactivator (tTA) was generated that is constitutively expressed in HeLa cells. This transactivator stimulates transcription from a minimal promoter sequence derived from the human cytomegalovirus promoter IE combined with tet operator sequences. Upon integration of a luciferase gene controlled by a tTA-dependent promoter into a tTA-producing HeLa cell line, high levels of luciferase expression were monitored. These activities are sensitive to tetracycline. Depending on the concentration of the antibiotic in the culture medium (0-1 microgram/ml), the luciferase activity can be regulated over up to five orders of magnitude. Thus, the system not only allows differential control of the activity of an individual gene in mammalian cells but also is suitable for creation of "on/off" situations for such genes in a reversible way.

Based upon observations on 48 cases of retinoblastoma and published reports, the hypothesis is developed that retinoblastoma is a cancer caused by two mutational events. In the dominantly inherited form, one mutation is inherited via the germinal cells and the second occurs in somatic cells. In the nonhereditary form, both mutations occur in somatic cells. The second mutation produces an average of three retinoblastomas per individual inheriting the first mutation. Using Poisson statistics, one can calculate that this number (three) can explain the occasional gene carrier who gets no tumor, those who develop only unilateral tumors, and those who develop bilateral tumors, as well as explaining instances of multiple tumors in one eye. This value for the mean number of tumors occurring in genetic carriers may be used to estimate the mutation rate for each mutation. The germinal and somatic rates for the first, and the somatic rate for the second, mutation, are approximately equal. The germinal mutation may arise in some instances from a delayed mutation.