Effective treatment of metastatic cancers usually requires the use of toxic chemotherapy. In most cases, multiple drugs are used, as resistance to single agents occurs almost universally. For this reason, elucidation of mechanisms that confer simultaneous resistance to different drugs with different targets and chemical structures - multidrug resistance - has been a major goal of cancer biologists during the past 35 years. Here, we review the most common of these mechanisms, one that relies on drug efflux from cancer cells mediated by ATP-binding cassette (ABC) transporters. We describe various approaches to combating multidrug-resistant cancer, including the development of drugs that engage, evade or exploit efflux by ABC transporters.

A critical barrier to entry into structure-based virtual screening is the lack of a suitable, easy to access database of purchasable compounds. We have therefore prepared a library of 727,842 molecules, each with 3D structure, using catalogs of compounds from vendors (the size of this library continues to grow). The molecules have been assigned biologically relevant protonation states and are annotated with properties such as molecular weight, calculated LogP, and number of rotatable bonds. Each molecule in the library contains vendor and purchasing information and is ready for docking using a number of popular docking programs. Within certain limits, the molecules are prepared in multiple protonation states and multiple tautomeric forms. In one format, multiple conformations are available for the molecules. This database is available for free download (http://zinc.docking.org) in several common file formats including SMILES, mol2, 3D SDF, and DOCK flexibase format. A Web-based query tool incorporating a molecular drawing interface enables the database to be searched and browsed and subsets to be created. Users can process their own molecules by uploading them to a server. Our hope is that this database will bring virtual screening libraries to a wide community of structural biologists and medicinal chemists.

BACKGROUND

Patients with type 2 diabetes are at high risk of fatal and non-fatal myocardial infarction and stroke. There is indirect evidence that agonists of peroxisome proliferator-activated receptor gamma (PPAR gamma) could reduce macrovascular complications. Our aim, therefore, was to ascertain whether pioglitazone reduces macrovascular morbidity and mortality in high-risk patients with type 2 diabetes.

METHODS

We did a prospective, randomised controlled trial in 5238 patients with type 2 diabetes who had evidence of macrovascular disease. We recruited patients from primary-care practices and hospitals. We assigned patients to oral pioglitazone titrated from 15 mg to 45 mg (n=2605) or matching placebo (n=2633), to be taken in addition to their glucose-lowering drugs and other medications. Our primary endpoint was the composite of all-cause mortality, non fatal myocardial infarction (including silent myocardial infarction), stroke, acute coronary syndrome, endovascular or surgical intervention in the coronary or leg arteries, and amputation above the ankle. Analysis was by intention to treat. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN NCT00174993.

RESULTS

Two patients were lost to follow-up, but were included in analyses. The average time of observation was 34.5 months. 514 of 2605 patients in the pioglitazone group and 572 of 2633 patients in the placebo group had at least one event in the primary composite endpoint (HR 0.90, 95% CI 0.80-1.02, p=0.095). The main secondary endpoint was the composite of all-cause mortality, non-fatal myocardial infarction, and stroke. 301 patients in the pioglitazone group and 358 in the placebo group reached this endpoint (0.84, 0.72-0.98, p=0.027). Overall safety and tolerability was good with no change in the safety profile of pioglitazone identified. 6% (149 of 2065) and 4% (108 of 2633) of those in the pioglitazone and placebo groups, respectively, were admitted to hospital with heart failure; mortality rates from heart failure did not differ between groups.

CONCLUSIONS

Pioglitazone reduces the composite of all-cause mortality, non-fatal myocardial infarction, and stroke in patients with type 2 diabetes who have a high risk of macrovascular events.

In recent years quality of life instruments have been featured as primary outcomes in many randomized trials. One of the challenges facing the investigator using such measures is determining the significance of any differences observed, and communicating that significance to clinicians who will be applying the trial results. We have developed an approach to elucidating the significance of changes in score in quality of life instruments by comparing them to global ratings of change. Using this approach we have established a plausible range within which the minimal clinically important difference (MCID) falls. In three studies in which instruments measuring dyspnea, fatigue, and emotional function in patients with chronic heart and lung disease were applied the MCID was represented by mean change in score of approximately 0.5 per item, when responses were presented on a seven point Likert scale. Furthermore, we have established ranges for changes in questionnaire scores that correspond to moderate and large changes in the domains of interest. This information will be useful in interpreting questionnaire scores, both in individuals and in groups of patients participating in controlled trials, and in the planning of new trials.

Adult marrow-derived Mesenchymal Stem Cells (MSCs) are capable of dividing and their progeny are further capable of differentiating into one of several mesenchymal phenotypes such as osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon-ligament fibroblasts, and adipocytes. In addition, these MSCs secrete a variety of cytokines and growth factors that have both paracrine and autocrine activities. These secreted bioactive factors suppress the local immune system, inhibit fibrosis (scar formation) and apoptosis, enhance angiogenesis, and stimulate mitosis and differentiation of tissue-intrinsic reparative or stem cells. These effects, which are referred to as trophic effects, are distinct from the direct differentiation of MSCs into repair tissue. Several studies which tested the use of MSCs in models of infarct (injured heart), stroke (brain), or meniscus regeneration models are reviewed within the context of MSC-mediated trophic effects in tissue repair.

This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.

Phosphatidylinositol (PtdIns) 3-kinase is an enzyme implicated in growth factor signal transduction by associating with receptor and nonreceptor tyrosine kinases, including the platelet-derived growth factor receptor. Inhibitors of PtdIns 3-kinase could potentially give a better understanding of the function and regulatory mechanisms of the enzyme. Quercetin, a naturally occurring bioflavinoid, was previously shown to inhibit PtdIns 3-kinase with an IC50 of 1.3 microgram/ml (3.8 microM); inhibition appeared to be directed at the ATP-binding site of the kinase. Analogs of quercetin were investigated as PtdIns 3-kinase inhibitors, with the most potent ones exhibiting IC50 values in the range of 1.7-8.4 micrograms/ml. In contrast, genistein, a potent tyrosine kinase inhibitor of the isoflavone class, did not inhibit PtdIns 3-kinase significantly (IC50>> 30 micrograms/ml). Since quercetin has also been shown to inhibit other PtdIns and protein kinases, other chromones were evaluated as inhibitors of PtdIns 3-kinase without affecting PtdIns 4-kinase or selected protein kinases. One such compound, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (also known as 2-(4-morpholinyl)-8-phenylchromone, LY294002), completely and specifically abolished PtdIns 3-kinase activity (IC50 = 0.43 microgram/ml; 1.40 microM) but did not inhibit PtdIns 4-kinase or tested protein and lipid kinases. Analogs of LY294002 demonstrated a very selective structure-activity relationship, with slight changes in structure causing marked decreases in inhibition. LY294002 was shown to completely abolish PtdIns 3-kinase activity in fMet-Leu-Phe-stimulated human neutrophils, as well as inhibit proliferation of smooth muscle cells in cultured rabbit aortic segments. Since PtdIns 3-kinase appears to be centrally involved with growth factor signal transduction, the development of specific inhibitors against the kinase may be beneficial in the treatment of proliferative diseases as well as in elucidating the biological role of the kinase in cellular proliferation and growth factor response.

The University of California Santa Cruz (UCSC) Table Browser (http://genome.ucsc.edu/cgi-bin/hgText) provides text-based access to a large collection of genome assemblies and annotation data stored in the Genome Browser Database. A flexible alternative to the graphical-based Genome Browser, this tool offers an enhanced level of query support that includes restrictions based on field values, free-form SQL queries and combined queries on multiple tables. Output can be filtered to restrict the fields and lines returned, and may be organized into one of several formats, including a simple tab- delimited file that can be loaded into a spreadsheet or database as well as advanced formats that may be uploaded into the Genome Browser as custom annotation tracks. The Table Browser User's Guide located on the UCSC website provides instructions and detailed examples for constructing queries and configuring output.

BACKGROUND

Depression is an important public-health problem, and one of the leading causes of disease burden worldwide. Depression is often comorbid with other chronic diseases and can worsen their associated health outcomes. Few studies have explored the effect of depression, alone or as a comorbidity, on overall health status.

METHODS

The WHO World Health Survey (WHS) studied adults aged 18 years and older to obtain data for health, health-related outcomes, and their determinants. Prevalence of depression in respondents based on ICD-10 criteria was estimated. Prevalence values for four chronic physical diseases--angina, arthritis, asthma, and diabetes--were also estimated using algorithms derived via a Diagnostic Item Probability Study. Mean health scores were constructed using factor analysis and compared across different disease states and demographic variables. The relation of these disease states to mean health scores was determined through regression modelling.

RESULTS

Observations were available for 245 404 participants from 60 countries in all regions of the world. Overall, 1-year prevalence for ICD-10 depressive episode alone was 3.2% (95% CI 3.0-3.5); for angina 4.5% (4.3-4.8); for arthritis 4.1% (3.8-4.3); for asthma 3.3% (2.9-3.6); and for diabetes 2.0% (1.8-2.2). An average of between 9.3% and 23.0% of participants with one or more chronic physical disease had comorbid depression. This result was significantly higher than the likelihood of having depression in the absence of a chronic physical disease (p<0.0001). After adjustment for socioeconomic factors and health conditions, depression had the largest effect on worsening mean health scores compared with the other chronic conditions. Consistently across countries and different demographic characteristics, respondents with depression comorbid with one or more chronic diseases had the worst health scores of all the disease states.

CONCLUSIONS

Depression produces the greatest decrement in health compared with the chronic diseases angina, arthritis, asthma, and diabetes. The comorbid state of depression incrementally worsens health compared with depression alone, with any of the chronic diseases alone, and with any combination of chronic diseases without depression. These results indicate the urgency of addressing depression as a public-health priority to reduce disease burden and disability, and to improve the overall health of populations.

p300/CBP is a transcriptional adaptor that integrates signals from many sequence-specific activators via direct interactions. Various cellular and viral factors target p300/CBP to modulate transcription and/or cell cycle progression. One such factor, the cellular p300/CBP associated factor (PCAF), possesses intrinsic histone acetyltransferase activity. Here, we demonstrate that p300/CBP is not only a transcriptional adaptor but also a histone acetyltransferase. p300/CBP represents a novel class of acetyltransferases in that it does not have the conserved motif found among various other acetyltransferases. p300/CBP acetylates all four core histones in nucleosomes. These observations suggest that p300/CBP acetylates nucleosomes in concert with PCAF.

MEME (Multiple EM for Motif Elicitation) is one of the most widely used tools for searching for novel 'signals' in sets of biological sequences. Applications include the discovery of new transcription factor binding sites and protein domains. MEME works by searching for repeated, ungapped sequence patterns that occur in the DNA or protein sequences provided by the user. Users can perform MEME searches via the web server hosted by the National Biomedical Computation Resource (http://meme.nbcr.net) and several mirror sites. Through the same web server, users can also access the Motif Alignment and Search Tool to search sequence databases for matches to motifs encoded in several popular formats. By clicking on buttons in the MEME output, users can compare the motifs discovered in their input sequences with databases of known motifs, search sequence databases for matches to the motifs and display the motifs in various formats. This article describes the freely accessible web server and its architecture, and discusses ways to use MEME effectively to find new sequence patterns in biological sequences and analyze their significance.

Some cases of Alzheimer's disease are inherited as an autosomal dominant trait. Genetic linkage studies have mapped a locus (AD3) associated with susceptibility to a very aggressive form of Alzheimer's disease to chromosome 14q24.3. We have defined a minimal cosegregating region containing the AD3 gene, and isolated at least 19 different transcripts encoded within this region. One of these transcripts (S182) corresponds to a novel gene whose product is predicted to contain multiple transmembrane domains and resembles an integral membrane protein. Five different missense mutations have been found that cosegregate with early-onset familial Alzheimer's disease. Because these changes occurred in conserved domains of this gene, and are not present in normal controls, they are likely to be causative of AD3.

Metabolism, like other aspects of life, involves tradeoffs. Oxidant by-products of normal metabolism cause extensive damage to DNA, protein, and lipid. We argue that this damage (the same as that produced by radiation) is a major contributor to aging and to degenerative diseases of aging such as cancer, cardiovascular disease, immune-system decline, brain dysfunction, and cataracts. Antioxidant defenses against this damage include ascorbate, tocopherol, and carotenoids. Dietary fruits and vegetables are the principal source of ascorbate and carotenoids and are one source of tocopherol. Low dietary intake of fruits and vegetables doubles the risk of most types of cancer as compared to high intake and also markedly increases the risk of heart disease and cataracts. Since only 9% of Americans eat the recommended five servings of fruits and vegetables per day, the opportunity for improving health by improving diet is great.

METHODS

The Oswestry Disability Index (ODI) has become one of the principal condition-specific outcome measures used in the management of spinal disorders. This review is based on publications using the ODI identified from the authors' personal databases, the Science Citation Index, and hand searches of Spine and current textbooks of spinal disorders.

OBJECTIVE

To review the versions of this instrument, document methods by which it has been validated, collate data from scores found in normal and back pain populations, provide curves for power calculations in studies using the ODI, and maintain the ODI as a gold standard outcome measure.

BACKGROUND

It has now been 20 years since its original publication. More than 200 citations exist in the Science Citation Index. The authors have a large correspondence file relating to the ODI, that is cited in most of the large textbooks related to spinal disorders.

METHODS

All the published versions of the questionnaire were identified. A systematic review of this literature was made. The various reports of validation were collated and related to a version.

RESULTS

Four versions of the ODI are available in English and nine in other languages. Some published versions contain misprints, and many omit the scoring system. At least 114 studies contain usable data. These data provide both validation and standards for other users and indicate the power of the instrument for detecting change in sample populations.

CONCLUSIONS

The ODI remains a valid and vigorous measure and has been a worthwhile outcome measure. The process of using the ODI is reviewed and should be the subject of further research. The receiver operating characteristics should be explored in a population with higher self-report disabilities. The behavior of the instrument is incompletely understood, particularly in sensitivity to real change.

The choice between life and death is one of the major events in regulation of the immune system. T cells that specifically recognize viral or bacterial antigens are selected to survive and proliferate in response to infection, whereas those that are self-reactive are eliminated via apoptosis. Even the survival of alloreactive T cells requires their proper costimulation and, when infection subsides, the activated T cells are eliminated. A major regulator of such life or death decisions is the transcription factor NF-kappaB. However, NF-kappaB cannot function alone. A variety of mechanisms exist to modulate its activity and thereby affect the ultimate outcome of a cell's fate.

BACKGROUND

It is not yet known whether DNA methylation levels can be used to accurately predict age across a broad spectrum of human tissues and cell types, nor whether the resulting age prediction is a biologically meaningful measure.

RESULTS

I developed a multi-tissue predictor of age that allows one to estimate the DNA methylation age of most tissues and cell types. The predictor, which is freely available, was developed using 8,000 samples from 82 Illumina DNA methylation array datasets, encompassing 51 healthy tissues and cell types. I found that DNA methylation age has the following properties: first, it is close to zero for embryonic and induced pluripotent stem cells; second, it correlates with cell passage number; third, it gives rise to a highly heritable measure of age acceleration; and, fourth, it is applicable to chimpanzee tissues. Analysis of 6,000 cancer samples from 32 datasets showed that all of the considered 20 cancer types exhibit significant age acceleration, with an average of 36 years. Low age-acceleration of cancer tissue is associated with a high number of somatic mutations and TP53 mutations, while mutations in steroid receptors greatly accelerate DNA methylation age in breast cancer. Finally, I characterize the 353 CpG sites that together form an aging clock in terms of chromatin states and tissue variance.

CONCLUSIONS

I propose that DNA methylation age measures the cumulative effect of an epigenetic maintenance system. This novel epigenetic clock can be used to address a host of questions in developmental biology, cancer and aging research.

Antigen stimulation of immune cells triggers Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels, promoting the immune response to pathogens by activating the transcription factor NFAT. We have previously shown that cells from patients with one form of hereditary severe combined immune deficiency (SCID) syndrome are defective in store-operated Ca2+ entry and CRAC channel function. Here we identify the genetic defect in these patients, using a combination of two unbiased genome-wide approaches: a modified linkage analysis with single-nucleotide polymorphism arrays, and a Drosophila RNA interference screen designed to identify regulators of store-operated Ca2+ entry and NFAT nuclear import. Both approaches converged on a novel protein that we call Orai1, which contains four putative transmembrane segments. The SCID patients are homozygous for a single missense mutation in ORAI1, and expression of wild-type Orai1 in SCID T cells restores store-operated Ca2+ influx and the CRAC current (I(CRAC)). We propose that Orai1 is an essential component or regulator of the CRAC channel complex.

To characterize proteins that bind to the immunoglobulin (Ig) heavy chain and the kappa light chain enhancers, an electrophoretic mobility shift assay with end-labeled DNA fragments was used. Three binding proteins have been found. One is NF-A, a factor found in all tested cell types that binds to the octamer sequence found upstream of all Ig variable region gene segments and to the same octamer in the heavy chain enhancer. The second, also ubiquitous, protein binds to a sequence in both the heavy chain and the kappa enhancers that was previously shown to be protected from methylation in vivo. Other closely related sites do not compete for this binding, implying a restriction enzyme-like binding specificity. The third protein binds to a sequence in the kappa enhancer (and to an identical sequence in the SV40 enhancer) and is restricted in its occurrence to B cells.

Vertebrates do not look like jellyfish because the bones of their skeletons are levers that allow movement and protect vital organs. Bones come in an enormous variety of shapes and sizes to accomplish these goals, but, with few exceptions, use one process--endochondral bone formation--to generate the skeleton. The past few years have seen an enormous increase in understanding of the signalling pathways and the transcription factors that control endochondral bone development.

COSMIC, the Catalogue Of Somatic Mutations In Cancer (http://cancer.sanger.ac.uk) is the world's largest and most comprehensive resource for exploring the impact of somatic mutations in human cancer. Our latest release (v70; Aug 2014) describes 2 002 811 coding point mutations in over one million tumor samples and across most human genes. To emphasize depth of knowledge on known cancer genes, mutation information is curated manually from the scientific literature, allowing very precise definitions of disease types and patient details. Combination of almost 20,000 published studies gives substantial resolution of how mutations and phenotypes relate in human cancer, providing insights into the stratification of mutations and biomarkers across cancer patient populations. Conversely, our curation of cancer genomes (over 12,000) emphasizes knowledge breadth, driving discovery of unrecognized cancer-driving hotspots and molecular targets. Our high-resolution curation approach is globally unique, giving substantial insight into molecular biomarkers in human oncology. In addition, COSMIC also details more than six million noncoding mutations, 10,534 gene fusions, 61,299 genome rearrangements, 695,504 abnormal copy number segments and 60,119,787 abnormal expression variants. All these types of somatic mutation are annotated to both the human genome and each affected coding gene, then correlated across disease and mutation types.

Tumor ascites fluids from guinea pigs, hamsters, and mice contain activity that rapidly increases microvascular permeability. Similar activity is also secreted by these tumor cells and a variety of other tumor cell lines in vitro. The permeability-increasing activity purified from either the culture medium or ascites fluid of one tumor, the guinea pig line 10 hepatocarcinoma, is a 34,000- to 42,000-dalton protein distinct from other known permeability factors.

BACKGROUND

Protein kinase B (PKB), also known as c-Akt, is activated rapidly when mammalian cells are stimulated with insulin and growth factors, and much of the current interest in this enzyme stems from the observation that it lies 'downstream' of phosphoinositide 3-kinase on intracellular signalling pathways. We recently showed that insulin or insulin-like growth factor 1 induce the phosphorylation of PKB at two residues, Thr308 and Ser473. The phosphorylation of both residues is required for maximal activation of PKB. The kinases that phosphorylate PKB are, however, unknown.

RESULTS

We have purified 500 000-fold from rabbit skeletal muscle extracts a protein kinase which phosphorylates PKBalpha at Thr308 and increases its activity over 30-fold. We tested the kinase in the presence of several inositol phospholipids and found that only low micromolar concentrations of the D enantiomers of either phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) or PtdIns(3,4)P2 were effective in potently activating the kinase, which has been named PtdIns(3,4,5)P3-dependent protein kinase-1 (PDK1). None of the inositol phospholipids tested activated or inhibited PKBalpha or induced its phosphorylation under the conditions used. PDK1 activity was not affected by wortmannin, indicating that it is not likely to be a member of the phosphoinositide 3-kinase family. CONLCUSIONS: PDK1 is likely to be one of the protein kinases that mediate the activation of PKB by insulin and growth factors. PDK1 may, therefore, play a key role in mediating many of the actions of the second messenger(s) PtdIns(3,4, 5)P3 and/or PtdIns(3,4)P2.

Tomato (Solanum lycopersicum) is a major crop plant and a model system for fruit development. Solanum is one of the largest angiosperm genera and includes annual and perennial plants from diverse habitats. Here we present a high-quality genome sequence of domesticated tomato, a draft sequence of its closest wild relative, Solanum pimpinellifolium, and compare them to each other and to the potato genome (Solanum tuberosum). The two tomato genomes show only 0.6% nucleotide divergence and signs of recent admixture, but show more than 8% divergence from potato, with nine large and several smaller inversions. In contrast to Arabidopsis, but similar to soybean, tomato and potato small RNAs map predominantly to gene-rich chromosomal regions, including gene promoters. The Solanum lineage has experienced two consecutive genome triplications: one that is ancient and shared with rosids, and a more recent one. These triplications set the stage for the neofunctionalization of genes controlling fruit characteristics, such as colour and fleshiness.