Major difficulties arise when introducing evidence and clinical guidelines into routine daily practice. Data show that many patients do not receive appropriate care, or receive unnecessary or harmful care. Many approaches claim to offer solutions to this problem; which ones are as yet the most effective and efficient is unclear. We aim to provide an overview of present knowledge about initiatives to changing medical practice. Substantial evidence suggests that to change behaviour is possible, but this change generally requires comprehensive approaches at different levels (doctor, team practice, hospital, wider environment), tailored to specific settings and target groups. Plans for change should be based on characteristics of the evidence or guideline itself and barriers and facilitators to change. In general, evidence shows that none of the approaches for transferring evidence to practice is superior to all changes in all situations.

In this paper we demonstrate in the intact human the possibility of a non-invasive modulation of motor cortex excitability by the application of weak direct current through the scalp. Excitability changes of up to 40 %, revealed by transcranial magnetic stimulation, were accomplished and lasted for several minutes after the end of current stimulation. Excitation could be achieved selectively by anodal stimulation, and inhibition by cathodal stimulation. By varying the current intensity and duration, the strength and duration of the after-effects could be controlled. The effects were probably induced by modification of membrane polarisation. Functional alterations related to post-tetanic potentiation, short-term potentiation and processes similar to postexcitatory central inhibition are the likely candidates for the excitability changes after the end of stimulation. Transcranial electrical stimulation using weak current may thus be a promising tool to modulate cerebral excitability in a non-invasive, painless, reversible, selective and focal way.

In observational studies with exposures or treatments that vary over time, standard approaches for adjustment of confounding are biased when there exist time-dependent confounders that are also affected by previous treatment. This paper introduces marginal structural models, a new class of causal models that allow for improved adjustment of confounding in those situations. The parameters of a marginal structural model can be consistently estimated using a new class of estimators, the inverse-probability-of-treatment weighted estimators.

The evolution of marine microbes over billions of years predicts that the composition of microbial communities should be much greater than the published estimates of a few thousand distinct kinds of microbes per liter of seawater. By adopting a massively parallel tag sequencing strategy, we show that bacterial communities of deep water masses of the North Atlantic and diffuse flow hydrothermal vents are one to two orders of magnitude more complex than previously reported for any microbial environment. A relatively small number of different populations dominate all samples, but thousands of low-abundance populations account for most of the observed phylogenetic diversity. This "rare biosphere" is very ancient and may represent a nearly inexhaustible source of genomic innovation. Members of the rare biosphere are highly divergent from each other and, at different times in earth's history, may have had a profound impact on shaping planetary processes.

Most human diseases are complex multi-factorial diseases resulting from the combination of various genetic and environmental factors. In the KEGG database resource (http://www.genome.jp/kegg/), diseases are viewed as perturbed states of the molecular system, and drugs as perturbants to the molecular system. Disease information is computerized in two forms: pathway maps and gene/molecule lists. The KEGG PATHWAY database contains pathway maps for the molecular systems in both normal and perturbed states. In the KEGG DISEASE database, each disease is represented by a list of known disease genes, any known environmental factors at the molecular level, diagnostic markers and therapeutic drugs, which may reflect the underlying molecular system. The KEGG DRUG database contains chemical structures and/or chemical components of all drugs in Japan, including crude drugs and TCM (Traditional Chinese Medicine) formulas, and drugs in the USA and Europe. This database also captures knowledge about two types of molecular networks: the interaction network with target molecules, metabolizing enzymes, other drugs, etc. and the chemical structure transformation network in the history of drug development. The new disease/drug information resource named KEGG MEDICUS can be used as a reference knowledge base for computational analysis of molecular networks, especially, by integrating large-scale experimental datasets.

The cloning of genes encoding mammalian DNA binding transcription factors for RNA polymerase II has provided the opportunity to analyze the structure and function of these proteins. This review summarizes recent studies that define structural domains for DNA binding and transcriptional activation functions in sequence-specific transcription factors. The mechanisms by which these factors may activate transcriptional initiation and by which they may be regulated to achieve differential gene expression are also discussed.

The intracellular storage and utilization of lipids are critical to maintain cellular energy homeostasis. During nutrient deprivation, cellular lipids stored as triglycerides in lipid droplets are hydrolysed into fatty acids for energy. A second cellular response to starvation is the induction of autophagy, which delivers intracellular proteins and organelles sequestered in double-membrane vesicles (autophagosomes) to lysosomes for degradation and use as an energy source. Lipolysis and autophagy share similarities in regulation and function but are not known to be interrelated. Here we show a previously unknown function for autophagy in regulating intracellular lipid stores (macrolipophagy). Lipid droplets and autophagic components associated during nutrient deprivation, and inhibition of autophagy in cultured hepatocytes and mouse liver increased triglyceride storage in lipid droplets. This study identifies a critical function for autophagy in lipid metabolism that could have important implications for human diseases with lipid over-accumulation such as those that comprise the metabolic syndrome.

Although vertebrate DNA is generally depleted in the dinucleotide CpG, it has recently been shown that some vertebrate genes contain CpG islands, regions of DNA with a high G+C content and a high frequency of CpG dinucleotides relative to the bulk genome. In this study, a large number of sequences of vertebrate genes were screened for the presence of CpG islands. Each CpG island was then analysed in terms of length, nucleotide composition, frequency of CpG dinucleotides, and location relative to the transcription unit of the associated gene. CpG islands were associated with the 5' ends of all housekeeping genes and many tissue-specific genes, and with the 3' ends of some tissue-specific genes. A few genes contained both 5' and 3' CpG islands, separated by several thousand base-pairs of CpG-depleted DNA. The 5' CpG islands extended through 5'-flanking DNA, exons and introns, whereas most of the 3' CpG islands appeared to be associated with exons. CpG islands were generally found in the same position relative to the transcription unit of equivalent genes in different species, with some notable exceptions. The locations of G/C boxes, composed of the sequence GGGCGG or its reverse complement CCGCCC, were investigated relative to the location of CpG islands. G/C boxes were found to be rare in CpG-depleted DNA and plentiful in CpG islands, where they occurred in 3' CpG islands, as well as in 5' CpG islands associated with tissue-specific and housekeeping genes. G/C boxes were located both upstream and downstream from the transcription start site of genes with 5' CpG islands. Thus, G/C boxes appeared to be a feature of CpG islands in general, rather than a feature of the promoter region of housekeeping genes. Two theories for the maintenance of a high frequency of CpG dinucleotides in CpG islands were tested: that CpG islands in methylated genomes are maintained, despite a tendency for 5mCpG to mutate by deamination to TpG+CpA, by the structural stability of a high G+C content alone, and that CpG islands associated with exons result from some selective importance of the arginine codon CGX. Neither of these theories could account for the distribution of CpG dinucleotides in the sequences analysed. Possible functions of CpG islands in transcriptional and post-transcriptional regulation of gene expression were discussed, and were related to theories for the maintenance of CpG islands as "methylation-free zones" in germline DNA.

Invasive candidiasis (IC) is a leading cause of mycosis-associated mortality in the United States. We examined data from the National Center for Health Statistics and reviewed recent literature in order to update the epidemiology of IC. IC-associated mortality has remained stable, at approximately 0.4 deaths per 100,000 population, since 1997, while mortality associated with invasive aspergillosis has continued to decline. Candida albicans remains the predominant cause of IC, accounting for over half of all cases, but Candida glabrata has emerged as the second most common cause of IC in the United States, and several less common Candida species may be emerging, some of which can exhibit resistance to triazoles and/or amphotericin B. Crude and attributable rates of mortality due to IC remain unacceptably high and unchanged for the past 2 decades. Nonpharmacologic preventive strategies should be emphasized, including hand hygiene; appropriate use, placement, and care of central venous catheters; and prudent use of antimicrobial therapy. Given that delays in appropriate antifungal therapy are associated with increased mortality, improved use of early empirical, preemptive, and prophylactic therapies should also help reduce IC-associated mortality. Several studies have now identified important variables that can be used to predict risk of IC and to help guide preventive strategies such as antifungal prophylaxis and early empirical therapy. However, improved non-culture-based diagnostics are needed to expand the potential for preemptive (or early directed) therapy. Further research to improve diagnostic, preventive, and therapeutic strategies is necessary to reduce the considerable morbidity and mortality associated with IC.

T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Recently, a clearer picture of the functional and phenotypic profile of exhausted T cells has emerged and T cell exhaustion has been defined in many experimental and clinical settings. Although the pathways involved remain to be fully defined, advances in the molecular delineation of T cell exhaustion are clarifying the underlying causes of this state of differentiation and also suggest promising therapeutic opportunities.

DNA damage is a relatively common event in the life of a cell and may lead to mutation, cancer, and cellular or organismic death. Damage to DNA induces several cellular responses that enable the cell either to eliminate or cope with the damage or to activate a programmed cell death process, presumably to eliminate cells with potentially catastrophic mutations. These DNA damage response reactions include: (a) removal of DNA damage and restoration of the continuity of the DNA duplex; (b) activation of a DNA damage checkpoint, which arrests cell cycle progression so as to allow for repair and prevention of the transmission of damaged or incompletely replicated chromosomes; (c) transcriptional response, which causes changes in the transcription profile that may be beneficial to the cell; and (d) apoptosis, which eliminates heavily damaged or seriously deregulated cells. DNA repair mechanisms include direct repair, base excision repair, nucleotide excision repair, double-strand break repair, and cross-link repair. The DNA damage checkpoints employ damage sensor proteins, such as ATM, ATR, the Rad17-RFC complex, and the 9-1-1 complex, to detect DNA damage and to initiate signal transduction cascades that employ Chk1 and Chk2 Ser/Thr kinases and Cdc25 phosphatases. The signal transducers activate p53 and inactivate cyclin-dependent kinases to inhibit cell cycle progression from G1 to S (the G1/S checkpoint), DNA replication (the intra-S checkpoint), or G2 to mitosis (the G2/M checkpoint). In this review the molecular mechanisms of DNA repair and the DNA damage checkpoints in mammalian cells are analyzed.

The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70-80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)-raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes.

The proportion of child deaths that occurs in the neonatal period (38% in 2000) is increasing, and the Millennium Development Goal for child survival cannot be met without substantial reductions in neonatal mortality. Every year an estimated 4 million babies die in the first 4 weeks of life (the neonatal period). A similar number are stillborn, and 0.5 million mothers die from pregnancy-related causes. Three-quarters of neonatal deaths happen in the first week--the highest risk of death is on the first day of life. Almost all (99%) neonatal deaths arise in low-income and middle-income countries, yet most epidemiological and other research focuses on the 1% of deaths in rich countries. The highest numbers of neonatal deaths are in south-central Asian countries and the highest rates are generally in sub-Saharan Africa. The countries in these regions (with some exceptions) have made little progress in reducing such deaths in the past 10-15 years. Globally, the main direct causes of neonatal death are estimated to be preterm birth (28%), severe infections (26%), and asphyxia (23%). Neonatal tetanus accounts for a smaller proportion of deaths (7%), but is easily preventable. Low birthweight is an important indirect cause of death. Maternal complications in labour carry a high risk of neonatal death, and poverty is strongly associated with an increased risk. Preventing deaths in newborn babies has not been a focus of child survival or safe motherhood programmes. While we neglect these challenges, 450 newborn children die every hour, mainly from preventable causes, which is unconscionable in the 21st century.

In the last two years the Pfam database (http://pfam.xfam.org) has undergone a substantial reorganisation to reduce the effort involved in making a release, thereby permitting more frequent releases. Arguably the most significant of these changes is that Pfam is now primarily based on the UniProtKB reference proteomes, with the counts of matched sequences and species reported on the website restricted to this smaller set. Building families on reference proteomes sequences brings greater stability, which decreases the amount of manual curation required to maintain them. It also reduces the number of sequences displayed on the website, whilst still providing access to many important model organisms. Matches to the full UniProtKB database are, however, still available and Pfam annotations for individual UniProtKB sequences can still be retrieved. Some Pfam entries (1.6%) which have no matches to reference proteomes remain; we are working with UniProt to see if sequences from them can be incorporated into reference proteomes. Pfam-B, the automatically-generated supplement to Pfam, has been removed. The current release (Pfam 29.0) includes 16 295 entries and 559 clans. The facility to view the relationship between families within a clan has been improved by the introduction of a new tool.

Exercise benefits a variety of organ systems in mammals, and some of the best-recognized effects of exercise on muscle are mediated by the transcriptional co-activator PPAR-γ co-activator-1 α (PGC1-α). Here we show in mouse that PGC1-α expression in muscle stimulates an increase in expression of FNDC5, a membrane protein that is cleaved and secreted as a newly identified hormone, irisin. Irisin acts on white adipose cells in culture and in vivo to stimulate UCP1 expression and a broad program of brown-fat-like development. Irisin is induced with exercise in mice and humans, and mildly increased irisin levels in the blood cause an increase in energy expenditure in mice with no changes in movement or food intake. This results in improvements in obesity and glucose homeostasis. Irisin could be therapeutic for human metabolic disease and other disorders that are improved with exercise.

A universal method to detect and determine siderophores was developed by using their high affinity for iron(III). The ternary complex chrome azurol S/iron(III)/hexadecyltrimethylammonium bromide, with an extinction coefficient of approximately 100,000 M-1 cm-1 at 630 nm, serves as an indicator. When a strong chelator removes the iron from the dye, its color turns from blue to orange. Because of the high sensitivity, determination of siderophores in solution and their characterization by paper electrophoresis chromatography can be performed directly on supernatants of culture fluids. The method is also applicable to agar plates. Orange halos around the colonies on blue agar are indicative of siderophore excretion. It was demonstrated with Escherichia coli strains that biosynthetic, transport, and regulatory mutations in the enterobactin system are clearly distinguishable. The method was successfully used to screen mutants in the iron uptake system of two Rhizobium meliloti strains, DM5 and 1021.

Rapid advances in sequencing technology have changed the experimental landscape of microbial ecology. In the last 10 years, the field has moved from sequencing hundreds of 16S rRNA gene fragments per study using clone libraries to the sequencing of millions of fragments per study using next-generation sequencing technologies from 454 and Illumina. As these technologies advance, it is critical to assess the strengths, weaknesses, and overall suitability of these platforms for the interrogation of microbial communities. Here, we present an improved method for sequencing variable regions within the 16S rRNA gene using Illumina's MiSeq platform, which is currently capable of producing paired 250-nucleotide reads. We evaluated three overlapping regions of the 16S rRNA gene that vary in length (i.e., V34, V4, and V45) by resequencing a mock community and natural samples from human feces, mouse feces, and soil. By titrating the concentration of 16S rRNA gene amplicons applied to the flow cell and using a quality score-based approach to correct discrepancies between reads used to construct contigs, we were able to reduce error rates by as much as two orders of magnitude. Finally, we reprocessed samples from a previous study to demonstrate that large numbers of samples could be multiplexed and sequenced in parallel with shotgun metagenomes. These analyses demonstrate that our approach can provide data that are at least as good as that generated by the 454 platform while providing considerably higher sequencing coverage for a fraction of the cost.

Although the existence of mammary stem cells has been suggested by serial transplantation studies in mice, their identification has been hindered by the lack of specific surface markers, and by the absence of suitable in vitro assays for testing stem cell properties: self-renewal and ability to generate differentiated progeny. We have developed an in vitro cultivation system that allows for propagation of human mammary epithelial cells (HMECs) in an undifferentiated state, based on their ability to proliferate in suspension, as nonadherent mammospheres. We demonstrate that nonadherent mammospheres are enriched in early progenitor/stem cells and able to differentiate along all three mammary epithelial lineages and to clonally generate complex functional structures in reconstituted 3D culture systems. Gene expression analysis of cells isolated from nonadherent mammospheres revealed overlapping genetic programs with other stem and progenitor cells and identified new markers that may be useful in the identification of mammary stem cells. The isolation and characterization of these stem cells should help elucidate the molecular pathways that govern normal mammary development and carcinogenesis.

Angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signalling pathways are affording demonstrable therapeutic efficacy in mouse models of cancer and in an increasing number of human cancers. However, in both preclinical and clinical settings, the benefits are at best transitory and are followed by a restoration of tumour growth and progression. Emerging data support a proposition that two modes of unconventional resistance underlie such results: evasive resistance, an adaptation to circumvent the specific angiogenic blockade; and intrinsic or pre-existing indifference. Multiple mechanisms can be invoked in different tumour contexts to manifest both evasive and intrinsic resistance, motivating assessment of their prevalence and importance and in turn the design of pharmacological strategies that confer enduring anti-angiogenic therapies.

BACKGROUND

Cardiac arrest outside the hospital is common and has a poor outcome. Studies in laboratory animals suggest that hypothermia induced shortly after the restoration of spontaneous circulation may improve neurologic outcome, but there have been no conclusive studies in humans. In a randomized, controlled trial, we compared the effects of moderate hypothermia and normothermia in patients who remained unconscious after resuscitation from out-of-hospital cardiac arrest.

METHODS

The study subjects were 77 patients who were randomly assigned to treatment with hypothermia (with the core body temperature reduced to 33 degrees C within 2 hours after the return of spontaneous circulation and maintained at that temperature for 12 hours) or normothermia. The primary outcome measure was survival to hospital discharge with sufficiently good neurologic function to be discharged to home or to a rehabilitation facility.

RESULTS

The demographic characteristics of the patients were similar in the hypothermia and normothermia groups. Twenty-one of the 43 patients treated with hypothermia (49 percent) survived and had a good outcome--that is, they were discharged home or to a rehabilitation facility--as compared with 9 of the 34 treated with normothermia (26 percent, P=0.046). After adjustment for base-line differences in age and time from collapse to the return of spontaneous circulation, the odds ratio for a good outcome with hypothermia as compared with normothermia was 5.25 (95 percent confidence interval, 1.47 to 18.76; P=0.011). Hypothermia was associated with a lower cardiac index, higher systemic vascular resistance, and hyperglycemia. There was no difference in the frequency of adverse events.

CONCLUSIONS

Our preliminary observations suggest that treatment with moderate hypothermia appears to improve outcomes in patients with coma after resuscitation from out-of-hospital cardiac arrest.

Currently, the most widely used criteria for assessing response to therapy in high-grade gliomas are based on two-dimensional tumor measurements on computed tomography (CT) or magnetic resonance imaging (MRI), in conjunction with clinical assessment and corticosteroid dose (the Macdonald Criteria). It is increasingly apparent that there are significant limitations to these criteria, which only address the contrast-enhancing component of the tumor. For example, chemoradiotherapy for newly diagnosed glioblastomas results in transient increase in tumor enhancement (pseudoprogression) in 20% to 30% of patients, which is difficult to differentiate from true tumor progression. Antiangiogenic agents produce high radiographic response rates, as defined by a rapid decrease in contrast enhancement on CT/MRI that occurs within days of initiation of treatment and that is partly a result of reduced vascular permeability to contrast agents rather than a true antitumor effect. In addition, a subset of patients treated with antiangiogenic agents develop tumor recurrence characterized by an increase in the nonenhancing component depicted on T2-weighted/fluid-attenuated inversion recovery sequences. The recognition that contrast enhancement is nonspecific and may not always be a true surrogate of tumor response and the need to account for the nonenhancing component of the tumor mandate that new criteria be developed and validated to permit accurate assessment of the efficacy of novel therapies. The Response Assessment in Neuro-Oncology Working Group is an international effort to develop new standardized response criteria for clinical trials in brain tumors. In this proposal, we present the recommendations for updated response criteria for high-grade gliomas.

Comparative modeling predicts the three-dimensional structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. The number of protein sequences that can be modeled and the accuracy of the predictions are increasing steadily because of the growth in the number of known protein structures and because of the improvements in the modeling software. Further advances are necessary in recognizing weak sequence-structure similarities, aligning sequences with structures, modeling of rigid body shifts, distortions, loops and side chains, as well as detecting errors in a model. Despite these problems, it is currently possible to model with useful accuracy significant parts of approximately one third of all known protein sequences. The use of individual comparative models in biology is already rewarding and increasingly widespread. A major new challenge for comparative modeling is the integration of it with the torrents of data from genome sequencing projects as well as from functional and structural genomics. In particular, there is a need to develop an automated, rapid, robust, sensitive, and accurate comparative modeling pipeline applicable to whole genomes. Such large-scale modeling is likely to encourage new kinds of applications for the many resulting models, based on their large number and completeness at the level of the family, organism, or functional network.

We describe the results of a genome-wide analysis of human cells that suggests that most protein-coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. We found that nucleosomes with H3K4me3 and H3K9,14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein-coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full-length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at postinitiation steps. Genes encoding most developmental regulators fall into this group. Our results also identify a class of genes that are excluded from experiencing transcription initiation, at which mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells.

BACKGROUND

Since the inception of the GO annotation project, a variety of tools have been developed that support exploring and searching the GO database. In particular, a variety of tools that perform GO enrichment analysis are currently available. Most of these tools require as input a target set of genes and a background set and seek enrichment in the target set compared to the background set. A few tools also exist that support analyzing ranked lists. The latter typically rely on simulations or on union-bound correction for assigning statistical significance to the results.

RESULTS

GOrilla is a web-based application that identifies enriched GO terms in ranked lists of genes, without requiring the user to provide explicit target and background sets. This is particularly useful in many typical cases where genomic data may be naturally represented as a ranked list of genes (e.g. by level of expression or of differential expression). GOrilla employs a flexible threshold statistical approach to discover GO terms that are significantly enriched at the top of a ranked gene list. Building on a complete theoretical characterization of the underlying distribution, called mHG, GOrilla computes an exact p-value for the observed enrichment, taking threshold multiple testing into account without the need for simulations. This enables rigorous statistical analysis of thousand of genes and thousands of GO terms in order of seconds. The output of the enrichment analysis is visualized as a hierarchical structure, providing a clear view of the relations between enriched GO terms.

CONCLUSIONS

GOrilla is an efficient GO analysis tool with unique features that make a useful addition to the existing repertoire of GO enrichment tools. GOrilla's unique features and advantages over other threshold free enrichment tools include rigorous statistics, fast running time and an effective graphical representation. GOrilla is publicly available at: http://cbl-gorilla.cs.technion.ac.il