Question: Emodin forms hydrogen bonds with residues involved in PGAM1 substrate binding.

Tyr26 phosphorylation of PGAM1 provides a metabolic advantage to tumours by stabilizing the active conformation
How oncogenic signalling coordinates glycolysis and anabolic biosynthesis in cancer cells remains unclear. We recently reported that the glycolytic enzyme phosphoglycerate mutase 1 (PGAM1) regulates anabolic biosynthesis by controlling intracellular levels of its substrate 3-phosphoglycerate and product 2-phosphoglycerate. Here we report a novel mechanism in which Y26 phosphorylation enhances PGAM1 activation through release of inhibitory E19 that blocks the active site, stabilising cofactor 2,3-bisphosphoglycerate binding and H11 phosphorylation. We also report the crystal structure of H11-phosphorylated PGAM1 and find that phospho-H11 activates PGAM1 at least in part by promoting substrate 3-phosphoglycerate binding. Moreover, Y26 phosphorylation of PGAM1 is common in human cancer cells and contributes to regulation of 3-phosphoglycerate and 2-phosphoglycerate levels, promoting cancer cell proliferation and tumour growth. As PGAM1 is a negative transcriptional target of TP53, and is therefore commonly upregulated in human cancers, these findings suggest that Y26 phosphorylation represents an additional acute mechanism underlying phosphoglycerate mutase 1 upregulation.

High-throughput parallel proteogenomics: a bacterial case study.
In recent years, a new paradigm for genome annotation has emerged, termed "proteogenomics," that leverages peptide MS to annotate a genome. This is achieved by mapping peptides to a six-frame translation of a genome, including available splice databases, which may suggest refinements to gene models. Using this approach, it is possible to refine gene regions such as exon boundaries, novel genes, gene boundaries, frame shifts, reverse strands, translated UTRs, and novel splice junctions. One of the challenges of proteogenomics is how best to (1) tackle assigning confidence to any resulting annotation and (2) apply these gene model refinements, either through manual annotation or through an automated process via training gene prediction tools. This is not a straightforward process, as many gene prediction tools have their defined suitability for niche genomes (either eukaryotic or prokaryotic) trained on and refined with model organisms such as Arabidopsis thaliana and Escherichia coli, and varying degrees of features that can leverage the use of external evidence. In this study, we outline a suitable approach toward preprocessing mass spectra and optimizing the MS/MS search for a given dataset. We also discuss future challenges, which continue to pose a problem in the field of proteogenomics, and better strategies to successfully tackle them with, using existing tools. We use Bradyrhizobium diazoefficiens (Nitrogen-fixing bacteria), with a 9.1 Mb genome as a case study, utilizing the latest in second-generation proteogenomics tools with multiple gene models for cross-validation of proteogenomics annotations.

Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration
Calorie restriction (CR) extends lifespan in a wide spectrum of organisms and is the only regimen known to lengthen the lifespan of mammals. We established a model of CR in budding yeast Saccharomyces cerevisiae. In this system, lifespan can be extended by limiting glucose or by reducing the activity of the glucose-sensing cyclic-AMP-dependent kinase (PKA). Lifespan extension in a mutant with reduced PKA activity requires Sir2 and NAD (nicotinamide adenine dinucleotide). In this study we explore how CR activates Sir2 to extend lifespan. Here we show that the shunting of carbon metabolism toward the mitochondrial tricarboxylic acid cycle and the concomitant increase in respiration play a central part in this process. We discuss how this metabolic strategy may apply to CR in animals.

Epigenetic instability in ES cells and cloned mice.
Cloning by nuclear transfer (NT) is an inefficient process in which most clones die before birth and survivors often display growth abnormalities. In an effort to correlate gene expression with survival and fetal overgrowth, we have examined imprinted gene expression in both mice cloned by nuclear transfer and in the embryonic stem (ES) cell donor populations from which they were derived. The epigenetic state of the ES cell genome was found to be extremely unstable. Similarly, variation in imprinted gene expression was observed in most cloned mice, even in those derived from ES cells of the same subclone. Many of the animals survived to adulthood despite widespread gene dysregulation, indicating that mammalian development may be rather tolerant to epigenetic aberrations of the genome. These data imply that even apparently normal cloned animals may have subtle abnormalities in gene expression.

Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides.
Among the pleiotropic effects of aminoglycosides, their irreversible uptake and their blockade of initiating ribosomes have appeared to explain their bactericidal action, while the contributions of translational misreading and membrane damage and the mechanism of that damage have remained uncertain. We now present evidence that incorporation of misread proteins into the membrane can account for the membrane damage. The bactericidal action thus appears to result from the following sequence, in which each step is essential: slight initial entry of the antibiotic; interaction with chain-elongating ribosomes, resulting in misreading; incorporation of misread protein into the membrane, creating abnormal channels; increased (and irreversible) entry through these channels, and hence increased misreading and formation of channels; and, finally, blockade of initiating ribosomes. This mechanism can account for several previously unexplained observations: that streptomycin uptake requires protein synthesis during, but not after, the lag before the membrane damage; that streptomycin-resistant cells, which fail to take up streptomycin, can do so after treatment by another aminoglycoside; and that puromycin at moderate concentrations accelerates streptomycin uptake, while high concentrations (which release shorter chains) prevent it. In addition, puromycin, prematurely releasing polypeptides of normal sequence, also evidently creates channels, since it is reported to promote streptomycin uptake even in streptomycin-resistant cells. These findings imply that normal membrane proteins must be selected not only for a hydrophobic anchoring surface, but also for a tight fit in the membrane.

Proteomic analysis reveals Warburg effect and anomalous metabolism of glutamine in pancreatic cancer cells.
In this present work, we characterized the proteomes of pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and normal pancreatic duct cells by mass spectrometry using LTQ-Orbitrap and identified more than 1700 proteins from each sample. On the basis of the spectra count label-free quantification approach, we identified a large number of differentially expressed metabolic enzymes and proteins involved in cytoskeleton, cell adhesion, transport, transcription, translation, and cell proliferation as well. The data demonstrated that metabolic pathways were altered in PANC-1, consistent with the Warburg effect. In addition, the comparative MS analysis unveiled anomalous metabolism of glutamine, suggesting that glutamine was largely consumed as a nitrogen donor in nucleotide and amino acid biosynthesis in PANC-1. Our analysis provides a potentially comprehensive picture of metabolism in PANC-1, which may serve as the basis of new diagnostics and treatment of PDAC.

Accuracy of ICD-9-CM codes for identifying cardiovascular and stroke risk factors.
OBJECTIVES We sought to determine which ICD-9-CM codes in Medicare Part A data identify cardiovascular and stroke risk factors. DESIGN AND PARTICIPANTS This was a cross-sectional study comparing ICD-9-CM data to structured medical record review from 23,657 Medicare beneficiaries aged 20 to 105 years who had atrial fibrillation. MEASUREMENTS Quality improvement organizations used standardized abstraction instruments to determine the presence of 9 cardiovascular and stroke risk factors. Using the chart abstractions as the gold standard, we assessed the accuracy of ICD-9-CM codes to identify these risk factors. MAIN RESULTS ICD-9-CM codes for all risk factors had high specificity (>0.95) and low sensitivity (< or =0.76). The positive predictive values were greater than 0.95 for 5 common, chronic risk factors-coronary artery disease, stroke/transient ischemic attack, heart failure, diabetes, and hypertension. The sixth common risk factor, valvular heart disease, had a positive predictive value of 0.93. For all 6 common risk factors, negative predictive values ranged from 0.52 to 0.91. The rare risk factors-arterial peripheral embolus, intracranial hemorrhage, and deep venous thrombosis-had high negative predictive value (> or =0.98) but moderate positive predictive values (range, 0.54-0.77) in this population. CONCLUSIONS Using ICD-9-CM codes alone, heart failure, coronary artery disease, diabetes, hypertension, and stroke can be ruled in but not necessarily ruled out. Where feasible, review of additional data (eg, physician notes or imaging studies) should be used to confirm the diagnosis of valvular disease, arterial peripheral embolus, intracranial hemorrhage, and deep venous thrombosis.

Restless legs syndrome and attention-deficit/hyperactivity disorder: a review of the literature.
STUDY OBJECTIVE To review evidence on the association between restless legs syndrome (RLS) and attention-deficit/hyperactivity disorder (ADHD), to discuss the hypothetical mechanisms underlying this association, and to consider the potential interest for common pharmacologic treatments of RLS and ADHD when co-occurring. METHOD A PubMed search. RESULTS In clinical samples, up to 44% of subjects with ADHD have been found to have RLS or RLS symptoms, and up to 26% of subjects with RLS have been found to have ADHD or ADHD symptoms. Several mechanisms may explain this association. Sleep disruption associated with RLS might lead to inattentiveness, moodiness, and paradoxical overactivity. Diurnal manifestations of RLS, such as restlessness and inattention, might mimic ADHD symptoms. Alternatively, RLS might be comorbid with idiopathic ADHD. Subjects with RLS and a subset of subjects with ADHD might share a common dopamine dysfunction. Limited evidence suggests that some dopaminergic agents, such as levodopa/carbidopa, pergolide, and ropinirole, may be effective in children with RLS associated with ADHD symptoms. CONCLUSIONS Although still limited, evidence from clinical studies demonstrates an association between RLS and ADHD or ADHD symptoms. Further clinical studies using standard criteria and procedures are needed to better estimate the degree of association. Epidemiologic studies are required to assess the relationship between ADHD and RLS symptoms in nonclinical samples. Further investigations should address the mechanisms underlying the relationship between RLS and ADHD. Several dopaminergic agents seem to be promising treatment for RLS associated with ADHD symptoms. To date, however, the absence of randomized and blinded controlled studies does not allow evidence-based recommendations.

The emergence of order in the Drosophila pupal retina.
During pupation, long-range order is imposed on the autonomously developing ommatidia which compose the Drosophila eye. To accomplish this, eight additional cell types arise: the primary, secondary, and tertiary pigment cells, and the four cells that form the bristle. These cells form an interweaving lattice between ommatidia. The lattice is refined when excess cells are removed to bring neighboring ommatidia into register. Recent evidence suggests that in larval development, local contacts direct cell fate. The same appears to be true during pupal development: the contacts a cell makes predict the cell type it will become. Cells which contact the anterior or posterior cone cells in an ommatidium invariably become primary pigment cells. Cells which contact primary pigment cells from different ommatidia become secondary and tertiary pigment cells. Bristle development is in several ways distinct from ommatidial development. The four cells of each bristle group appear to be immediate descendents of a single founder cell. During their early differentiation, they do not make stereotyped contacts with surrounding ommatidial cells, but do make particular contacts within the bristle group. And unlike the surrounding ommatidia, differentiation of the bristles radiates from the center of the eye to the edges. As cells are removed during two stages of programmed cell death, the bristles are brought into their final position. When all cells in the lattice have achieved their final position, a second stage of retinal development begins as structures specific to each cell type are produced. This paper follows these various stages of pupal development, and suggests how local cell-cell contacts may produce the cells needed for a functional retina.

Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo
Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. However, the mechanisms behind these effects are unclear. Here we show that nicotine, a major constituent of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which in turn phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating its proteasome-dependent degradation. The nicotine-dependent reduction of MKP1 induces the aberrant activation of both p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. Phosphorylation of IRS1 leads to its degradation, protein kinase B inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results establish AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity.

Advantages and pitfalls of fructosamine and glycated albumin in the diagnosis and treatment of diabetes.
The efficient diagnosis and accurate monitoring of diabetic patients are cornerstones for reducing the risk of diabetic complications. The current diagnostic and prognostic strategies in diabetes are mainly based on two tests, plasma (or capillary) glucose and glycated hemoglobin (HbA1c). Nevertheless, these measures are not foolproof, and their clinical usefulness is biased by a number of clinical and analytical factors. The introduction of other indices of glucose homeostasis in clinical practice such as fructosamine and glycated albumin (GA) may be regarded as an attractive alternative, especially in patients in whom the measurement of HbA1c may be biased or even unreliable. These include patients with rapid changes of glucose homeostasis and larger glycemic excursions, and patients with red blood cell disorders and renal disease. According to available evidence, the overall diagnostic efficiency of GA seems superior to that of fructosamine throughout a broad range of clinical settings. The current method for measuring GA is also better standardized and less vulnerable to preanalytical variables than those used for assessing fructosamine. Additional advantages of GA over HbA1c are represented by lower reagent cost and being able to automate the GA analysis on many conventional laboratory instruments. Although further studies are needed to definitely establish that GA can complement or even replace conventional measures of glycemic control such as HbA1c, GA may help the clinical management of patients with diabetes in whom HbA1c values might be unreliable.

Instruction: Given a scientific claim, retrieve documents that support or refute the claim


Question: Energy balance requires hypothalamic glutamate neurotransmission.

Glutamate mediates the function of melanocortin receptor 4 on Sim1 neurons in body weight regulation.
The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing brain regions, which include the paraventricular nucleus of hypothalamus (PVH), represent key brain sites that mediate melanocortin action. We conditionally restored MC4R expression in Sim1 neurons in the background of Mc4r-null mice. The restoration dramatically reduced obesity in Mc4r-null mice. The anti-obesity effect was completely reversed by selective disruption of glutamate release from those same Sim1 neurons. The reversal was caused by lower energy expenditure and hyperphagia. Corroboratively, selective disruption of glutamate release from adult PVH neurons led to rapid obesity development via reduced energy expenditure and hyperphagia. Thus, this study establishes glutamate as the primary neurotransmitter that mediates MC4Rs on Sim1 neurons in body weight regulation.

Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes induced pluripotent stem cell reprogramming.
While most somatic cells undergoing induced pluripotent stem (iPS) cell reprogramming with Yamanaka factors accumulate at stable partially reprogrammed stages, the molecular mechanisms required to achieve full reprogramming are unknown. MicroRNAs (miRNAs) fine-tune mRNA translation and are implicated in reprogramming, but miRNA functional targets critical for complete iPS cell reprogramming remain elusive. We identified methyl-DNA binding domain protein 2 (MBD2) as an epigenetic suppressor, blocking full reprogramming of somatic to iPS cells through direct binding to NANOG promoter elements preventing transcriptional activation. When we overexpressed miR-302 cluster we observed a significant increase in conversion of partial to fully reprogrammed iPS cells by suppressing MBD2 expression, thereby increasing NANOG expression. Thus, expression of exogenous miR-302 cluster (without miR-367) is efficient in attaining a fully reprogrammed iPS state in partially reprogrammed cells by relieving MBD2-mediated inhibition of NANOG expression. Our studies provide a direct molecular mechanism involved in generating complete human iPS cell reprogramming to study disease pathogenesis, drug screening, and for potential cell-based therapies.

Full-length transcriptome assembly from RNA-Seq data without a reference genome.
Massively parallel sequencing of cDNA has enabled deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here we present the Trinity method for de novo assembly of full-length transcripts and evaluate it on samples from fission yeast, mouse and whitefly, whose reference genome is not yet available. By efficiently constructing and analyzing sets of de Bruijn graphs, Trinity fully reconstructs a large fraction of transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Compared with other de novo transcriptome assemblers, Trinity recovers more full-length transcripts across a broad range of expression levels, with a sensitivity similar to methods that rely on genome alignments. Our approach provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome.

Immune activation and HIV persistence: implications for curative approaches to HIV infection.
Despite complete or near-complete suppression of human immunodeficiency virus (HIV) replication with combination antiretroviral therapy, both HIV and chronic inflammation/immune dysfunction persist indefinitely. Untangling the association between the virus and the host immune environment during therapy might lead to novel interventions aimed at either curing the infection or preventing the development of inflammation-associated end-organ disease. Chronic inflammation and immune dysfunction might lead to HIV persistence by causing virus production, generating new target cells, enabling infecting of activated and resting target cells, altering the migration patterns of susceptible target cells, increasing the proliferation of infected cells, and preventing normal HIV-specific clearance mechanisms from function. Chronic HIV production or replication might contribute to persistent inflammation and immune dysfunction. The rapidly evolving data on these issues strongly suggest that a vicious cycle might exist in which HIV persistence causes inflammation that in turn contributes to HIV persistence.

Advantages and pitfalls of fructosamine and glycated albumin in the diagnosis and treatment of diabetes.
The efficient diagnosis and accurate monitoring of diabetic patients are cornerstones for reducing the risk of diabetic complications. The current diagnostic and prognostic strategies in diabetes are mainly based on two tests, plasma (or capillary) glucose and glycated hemoglobin (HbA1c). Nevertheless, these measures are not foolproof, and their clinical usefulness is biased by a number of clinical and analytical factors. The introduction of other indices of glucose homeostasis in clinical practice such as fructosamine and glycated albumin (GA) may be regarded as an attractive alternative, especially in patients in whom the measurement of HbA1c may be biased or even unreliable. These include patients with rapid changes of glucose homeostasis and larger glycemic excursions, and patients with red blood cell disorders and renal disease. According to available evidence, the overall diagnostic efficiency of GA seems superior to that of fructosamine throughout a broad range of clinical settings. The current method for measuring GA is also better standardized and less vulnerable to preanalytical variables than those used for assessing fructosamine. Additional advantages of GA over HbA1c are represented by lower reagent cost and being able to automate the GA analysis on many conventional laboratory instruments. Although further studies are needed to definitely establish that GA can complement or even replace conventional measures of glycemic control such as HbA1c, GA may help the clinical management of patients with diabetes in whom HbA1c values might be unreliable.

Population dynamics of a pathogen: the conundrum of vivax malaria
Building a mathematical model of population dynamics of pathogens within their host involves considerations of factors similar to those in ecology, as pathogens can prey on cells in the host. But within the multicellular host, attacked cell types are integrated with other cellular systems, which in turn intervene in the infection. For example, immune responses attempt to sense and then eliminate or contain pathogens, and homeostatic mechanisms try to compensate for cell loss. This review focuses on modeling applied to malarias, diseases caused by single-cell eukaryote parasites that infect red blood cells, with special concern given to vivax malaria, a disease often thought to be benign (if sometimes incapacitating) because the parasite only attacks a small proportion of red blood cells, the very youngest ones. However, I will use mathematical modeling to argue that depletion of this pool of red blood cells can be disastrous to the host if growth of the parasite is not vigorously check by host immune responses. Also, modeling can elucidate aspects of new field observations that indicate that vivax malaria is more dangerous than previously thought. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12551-010-0034-3) contains supplementary material, which is available to authorized users.

Immune reconstitution after cord blood transplantation: peculiarities, clinical implications and management strategies.
Umbilical cord blood (UCB) is now widely used as an alternative hematopoietic stem cell source for patients lacking closely matched related or unrelated adult donors. UCB transplantation has traditionally been associated with delayed engraftment, poor immune reconstitution and consequent increased risk of infection. More recent clinical studies, however, suggest that conditioning regimens and in particular the omission of in vivo T-cell depletion may play a crucial role in post-transplant T-cell expansion, facilitating a uniquely rapid immune recovery after UCB transplantation. The peculiar characteristics of UCB cells, the importance of thymic function and the role of conditioning regimens and graft-versus-host disease influencing immune reconstitution are described. The last part of the review reports available data on UCB, as well as third-party peripheral blood derived anti-viral cell therapy, which provides a novel approach to rescue UCB recipients with viral complications in the post-transplant period.

Triglyceride depletion of brown adipose tissue enables analysis of mitochondrial respiratory function in permeabilized biopsies.
The research on mitochondrial functions in adipocytes has increasingly evidenced that mitochondria plays an important role in the onset and/or progression of obesity and related pathologies. Mitochondrial function in brown adipose tissue (BAT) has been classically assessed by measuring either the levels/activity of mitochondrial enzymes, or the respiration in isolated mitochondria. Isolation of mitochondria is not advantageous because it demands significant time and amount of tissue and, as tissue homogenates, disrupts biochemical and physical connections of mitochondria within the cell. Here, we described a new and efficient protocol to analyze the mitochondrial respiratory states in BAT biopsies that relies on intracellular triglyceride depletion followed by tissue permeabilization. In addition to minimizing tissue requirements to ∼17 mg wet weight, the proposed protocol enabled analysis of all mitochondrial respiratory states, including phosphorylation (OXPHOS), no-phosphorylation (LEAK), and uncoupled (ETS) states, as well as the use of substrates for complex I, complex II, and cytochrome c; together, these features demonstrated mitochondrial integrity and validated the preparation efficacy. Therefore, the protocol described here increases the possibilities of answering physiological questions related to small BAT regions of human and animal models, which shall help to unravel the mechanisms that regulate mitochondrial function in health and disease.

Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL
The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.

Placebo-Controlled Trials and Active-Control Trials in the Evaluation of New Treatments. Part 1: Ethical and Scientific Issues
Placebo-controlled trials are used extensively in the development of new pharmaceuticals. They are sometimes challenged as unethical in settings in which patients could be treated with an existing therapy (1-7). The issues of when placebo controls are ethically acceptable and when they are scientifically necessary are important and worthy of discussion. The Ethics of Placebo Controls The Declaration of Helsinki The Declaration of Helsinki (8) is an international document that describes ethical principles for clinical investigation. Those who contend that placebo controls are unethical whenever known effective therapy exists for a condition usually cite the following sentence in the Declaration as support for that position: In any medical study, every patientincluding those of a control group, if anyshould be assured of the best proven diagnostic and therapeutic method. We believe that an interpretation of this sentence as barring placebo controls whenever an effective treatment exists is untenable. First, the requirement that all patients receive the best proven diagnostic and therapeutic method would bar not only placebo-controlled trials but also active-control and historically controlled trials. When effective treatment exists, the patient receiving the investigational treatment instead of the established therapy is clearly not getting the best proven treatment. Second, it does not seem reasonable to consider as equivalent all failures to use known effective therapy. Historically, concerns about placebo use have usually arisen in the context of serious illness. There is universal agreement that use of placebo or otherwise untreated controls is almost always unethical when therapy shown to improve survival or decrease serious morbidity is available. But in cases in which the treatment does not affect the patient's long-term health, an ethical imperative to use existing therapy is not plausible. Can it be, for example, that because topical minoxidil or oral finasteride can grow hair, a placebo-controlled trial of a new remedy for baldness is unethical? Is it really unethical to use placebos in short-term studies of drugs for allergic rhinitis, insomnia, anxiety, dermatoses, heartburn, or headaches in fully informed patients? We do not believe that there is a reasonable basis for arguing that such studies and many other placebo-controlled studies of symptom relief are unethical and that an informed patient cannot properly be asked to participate in them. Third, there is good reason to doubt that the cited phrase was intended to discourage placebo-controlled trials. The phrase under discussion was not part of the original 1964 Declaration but was added in 1975 to reinforce the idea that the physicianpatient relationship must be respected just as it would be in a purely therapeutic situation not involving research objectives (8). In the explanation accompanying the 1975 change, the issue of placebo-controlled trials was not even mentioned (9). The American Medical Association (10), the World Health Organization (11), and the Council for International Organizations of Medical Sciences (12) have rejected the position that the Declaration uniformly bars placebo-controlled trials when proven therapy is available. Informed Consent in Placebo-Controlled Trials Patients asked to participate in a placebo-controlled trial must be informed of the existence of any effective therapy, must be able to explore the consequences of deferring such therapy with the investigator, and must provide fully informed consent. Concern about whether consent to participate in trials is as informed as we would like to believe is valid, but these concerns apply as much to the patient's decision to forgo known effective treatment and risk exposure to a potentially ineffective or even harmful new agent in an active-control trial as to a decision to accept possible persistence of symptoms in a placebo-controlled trial. Thus, this problem is not unique to placebo-controlled trials. For the above reasons, we conclude that placebo-controlled trials may be ethically conducted even when effective therapy exists, as long as patients will not be harmed by participation and are fully informed about their alternatives. Although in many cases application of this standard will be fairly straightforward, in others it will not, and there may be debate about the consequences of deferring treatment (13). Assessment of Effectiveness with Active-Control Trials Clinical trials that, because of deficiencies in study design or conduct, are unlikely to provide scientifically valid and clinically meaningful results raise their own ethical concerns (12, 14). The remainder of this paper will address the inability of commonly proposed alternatives to placebo-controlled trials to evaluate the effectiveness of new treatments in many medical settings. Active-Control Equivalence Trials (Noninferiority Trials) The ability to conduct a placebo-controlled trial ethically in a given situation does not necessarily mean that placebo-controlled trials should be carried out when effective therapy exists. Patients and physicians might still prefer a trial in which every participant is given an active treatment. What remains to be examined is why placebo-controlled trials (or, more generally, trials intended to show an advantage of one treatment over another) are frequently needed to demonstrate the effectiveness of new treatments and often cannot be replaced by active-control trials showing that a new drug is equivalent or noninferior to a known effective agent. The limitations of active-control equivalence trials (ACETs) that are intended to show the effectiveness of a new drug have long been recognized and are well described (15-33) but are perhaps not as widely appreciated as they should be. A recent proposed international guideline on choice of control group addresses this issue in detail (33). The Fundamental Problem: Need for Assay Sensitivity There are two distinct ways to show that a new therapy is effective. One can show that the new therapy is superior to a control treatment, or one can show that the new therapy is equivalent to or not worse by some defined amount than a known effective treatment. Each method can be valid, but each requires entirely different inferential approaches. A well-designed study that shows superiority of a treatment to a control (placebo or active therapy) provides strong evidence of the effectiveness of the new treatment, limited only by the statistical uncertainty of the result. No information external to the trial is needed to support the conclusion of effectiveness. In contrast, a study that successfully shows equivalencethat is, little difference between a new drug and known active treatmentdoes not by itself demonstrate that the new treatment is effective. Equivalence could mean that the treatments were both effective in the study, but it could also mean that both treatments were ineffective in the study. To conclude from an ACET that a new treatment is effective on the basis of its similarity to the active control, one must make the critical (and untestable within the study) assumption that the active control had an effect in that particular study. In other words, one must assume that if a placebo group had been included, the placebo would have been inferior to the active control (15-33). Support for this assumption must come from sources external to the trial. Although it might appear reasonable to expect a known active agent to be superior to placebo in any given appropriately designed trial, experience has shown that this is not the case for many types of drugs. The ability of a study to distinguish between active and inactive treatments is termed assay sensitivity. If assay sensitivity cannot be assumed, then even if the new and standard treatments appear virtually identical and the confidence interval for their comparison is exquisitely narrow, the study cannot demonstrate effectiveness of the new drug. (Note that in practice, ACETs are not designed simply to show lack of a statistically significant difference between treatments. Rather, such trials are designed to show noninferioritythat the new treatment is not inferior to the control by more than a specified margin. This approach is described in the Appendix.) The best evidence that an active drug would have an effect superior to that of placebo in a given study would be a series of trials of similar design in which the active drug has reliably outperformed placebo. The ACET thus requires information external to the trial (the information about past placebo-controlled studies of the active control) to interpret the results. In this respect, an ACET is similar to a historically controlled trial. In some settings, such as highly responsive cancers, most infectious diseases, and some cardiovascular conditions, such external information is available and ACETs can and do provide a valid and reliable basis for evaluating new treatments. In many cases, however, the historically based assumption of assay sensitivity cannot be made; for many types of effective drugs, studies of apparently adequate size and design do not regularly distinguish drugs from placebo (16-18, 25, 34). More than 20 years ago, Lasagna (19) described this difficulty particularly well (reflecting long recognition of the problem among analgesiologists): a comparison between new drug and standard is convincing only when the new remedy is superior to standard treatment. If it is inferior, or even indistinguishable from a standard remedy, the results are not readily interpretable. In the absence of placebo controls, one does not know if the inferior new medicine has any efficacy at all, and equivalent performance may reflect simply a patient population that cannot distinguish between two active treatments that differ considerably from each other, or between active drug and placebo. Certain clinical conditions, such as seri

Multi-stage chemical carcinogenesis in mouse skin: Fundamentals and applications
For more than 60 years, the chemical induction of tumors in mouse skin has been used to study mechanisms of epithelial carcinogenesis and evaluate modifying factors. In the traditional two-stage skin carcinogenesis model, the initiation phase is accomplished by the application of a sub-carcinogenic dose of a carcinogen. Subsequently, tumor development is elicited by repeated treatment with a tumor-promoting agent. The initiation protocol can be completed within 1–3 h depending on the number of mice used; whereas the promotion phase requires twice weekly treatments (1–2 h) and once weekly tumor palpation (1–2 h) for the duration of the study. Using the protocol described here, a highly reproducible papilloma burden is expected within 10–20 weeks with progression of a portion of the tumors to squamous cell carcinomas within 20–50 weeks. In contrast to complete skin carcinogenesis, the two-stage model allows for greater yield of premalignant lesions, as well as separation of the initiation and promotion phases.

Instruction: Given a scientific claim, retrieve documents that support or refute the claim


Question: Energy balance requires hypothalamic glutamate neurotransmission.

Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone
AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons. Deletion of NMDARs from AgRP neurons markedly reduced weight, body fat and food intake whereas deletion from POMC neurons had no effect. Activation of AgRP neurons by fasting, as assessed by c-Fos, Agrp and Npy mRNA expression, AMPA receptor-mediated EPSCs, depolarization and firing rates, required NMDARs. Furthermore, AgRP but not POMC neurons have dendritic spines and increased glutamatergic input onto AgRP neurons caused by fasting was paralleled by an increase in spines, suggesting fasting induced synaptogenesis and spinogenesis. Thus glutamatergic synaptic transmission and its modulation by NMDARs play key roles in controlling AgRP neurons and determining the cellular and behavioral response to fasting.

β-Catenin Signaling and Roles in Liver Homeostasis, Injury, and Tumorigenesis.
β-catenin (encoded by CTNNB1) is a subunit of the cell surface cadherin protein complex that acts as an intracellular signal transducer in the WNT signaling pathway; alterations in its activity have been associated with the development of hepatocellular carcinoma and other liver diseases. Other than WNT, additional signaling pathways also can converge at β-catenin. β-catenin also interacts with transcription factors such as T-cell factor, forkhead box protein O, and hypoxia inducible factor 1α to regulate the expression of target genes. We discuss the role of β-catenin in metabolic zonation of the adult liver. β-catenin also regulates the expression of genes that control metabolism of glucose, nutrients, and xenobiotics; alterations in its activity may contribute to the pathogenesis of nonalcoholic steatohepatitis. Alterations in β-catenin signaling may lead to activation of hepatic stellate cells, which is required for fibrosis. Many hepatic tumors such as hepatocellular adenomas, hepatocellular cancers, and hepatoblastomas have mutations in CTNNB1 that result in constitutive activation of β-catenin, so this molecule could be a therapeutic target. We discuss how alterations in β-catenin activity contribute to liver disease and how these might be used in diagnosis and prognosis, as well as in the development of therapeutics.

Elevated expression of miR-24-3p is a potentially adverse prognostic factor in colorectal adenocarcinoma.
OBJECTIVES Several miRNAs are aberrantly expressed in cancer. miR-24-3p is involved in cancer-related cellular processes, including cell cycle control, cell growth, proliferation, and apoptosis. In this study, we examined the potential diagnostic and prognostic significance of miR-24-3p expression in colorectal adenocarcinoma. DESIGN AND METHODS Total RNA was isolated from 182 colorectal adenocarcinoma specimens and 86 paired non-cancerous colorectal mucosae. After polyadenylation of 2μg total RNA and reverse transcription into first-strand cDNA using an oligo-dT-adapter primer, miR-24-3p expression was quantified using an in-house-developed reverse-transcription real-time quantitative PCR method, based on the SYBR Green chemistry. SNORD43 (RNU43) was used as a reference gene. RESULTS miR-24-3p levels do not significantly differ between colorectal adenocarcinoma and non-cancerous colorectal mucosae. Thus, miR-24-3p expression cannot be used for diagnostic purposes. However, high miR-24-3p expression predicts poor disease-free survival (DFS) and overall survival (OS) of colorectal adenocarcinoma patients. Multivariate Cox regression analysis confirmed that miR-24-3p overexpression is a significant predictor of relapse in colorectal adenocarcinoma and that its prognostic significance is independent of other established prognostic factors and treatment of patients. Of note, miR-24-3p overexpression retains its rather unfavorable prognostic value in the subgroup of patients with advanced yet locally restricted colorectal adenocarcinoma (T3) and in those without distant metastasis (M0). Moreover, miR-24-3p overexpression is a potentially unfavorable prognosticator for patients who were not treated with radiotherapy. CONCLUSIONS Strong expression of miR-24-3p predicts poor DFS and OS of colorectal adenocarcinoma patients, independently of clinicopathological parameters that are currently used for prognosis in this human malignancy.

Integrin αvβ3/c-src “Oncogenic Unit” Promotes Anchorage-independence and Tumor Progression
Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin alpha(v)beta(3) is associated with progression of a variety of human tumors. Here we reveal a previously undescribed adhesion-independent role for integrin alpha(v)beta(3) in pancreatic cancer and other carcinomas. Specifically, alpha(v)beta(3) expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. These effects required recruitment of c-Src to the beta(3) integrin cytoplasmic tail, leading to c-Src activation, Crk-associated substrate (CAS) phosphorylation and tumor cell survival that, unexpectedly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Pharmacological blockade of c-Src kinase activity or decreased expression of endogenous alpha(v)beta(3) integrin or c-Src not only inhibited anchorage-independent growth but also suppressed metastasis in vivo, yet these manipulations did not affect tumor cell migration or invasion. These data define an unexpected role for an integrin as a mediator of anchorage independence, suggesting that an alpha(v)beta(3)-c-Src signaling module may account for the aggressive behavior of integrin alpha(v)beta(3)-expressing tumors in humans.

Radioiodine treatment of multinodular non-toxic goitre.
OBJECTIVE To investigate the long term effect of radioactive iodine on thyroid function and size in patients with non-toxic multinodular goitre. DESIGN Consecutive patients with multinodular non-toxic goitre selected for radioactive iodine treatment and followed for a minimum of 12 months (median 48 months) after an intended dose of 3.7 MBq/g thyroid tissue corrected to a 100% uptake of iodine-131 in 24 hours. PATIENTS 69 patients with a growing multinodular non-toxic goitre causing local compression symptoms or cosmetic inconveniences. The treatment was chosen because of a high operative risk, previous thyroidectomy, or refusal to be operated on. MAIN OUTCOME MEASUREMENTS Standard thyroid function variables and ultrasonically determined thyroid volume before treatment as well as 1, 2, 3, 6, and 12 months after treatment and then once a year. RESULTS 56 patients were treated with a single dose of 131I, 12 with two doses, and one with four doses. In 45 patients treated with one dose and remaining euthyroid the median thyroid volume was reduced from 73 (interquartile range 50-106) ml to 29 (23-48) ml at 24 months in the 39 patients in whom this was measured during follow up. The median reduction was 40 (22-48) ml (60% reduction, p < 0.0001), half of which occurred within three months. Patients treated with two doses as well as those developing hypothyroidism and hyperthyroidism had a significant reduction in thyroid volume. Eleven patients developed hypothyroidism (cumulative five year risk 22%, 95% confidence interval 4.8% to 38.4%). Side effects were few: three cases of hyperthyroidism and two cases of radiation thyroiditis. Only one patient was dissatisfied with the result; she was referred for operation six months after treatment. CONCLUSIONS A substantial reduction in thyroid volume accompanied by a low incidence of hypothyroidism and few side effects makes the use of radioactive iodine an attractive alternative to surgery in selected cases of non-toxic multinodular goitre.

Advances in techniques of testing mycobacterial drug sensitivity, and the use of sensitivity tests in tuberculosis control programmes.
In a paper arising out of an informal international consultation of specialists in the bacteriology of tuberculosis held in 1961, an attempt was made to formulate criteria, and specify technical procedures, for reliable tests of sensitivity (the absolute-concentration method, the resistance-ratio method and the proportion method) to the 3 main antituberculosis drugs (isoniazid, streptomycin and p-aminosalicylic acid). Seven years later, a further consultation was held to review the latest developments in the field and to suggest how sensitivity tests might be put to practical use in tuberculosis control programmes. The participants reached agreement on how to define drug sensitivity and resistance, and stressed the importance of using a discrimination approach to the calibration of sensitivity tests. Their views are contained in the present paper, which also includes descriptions of the sensitivity tests used by the Medical Research Council of Great Britain for first- and second-line drugs (minimal inhibitory concentration and resistance-ratio methods), the two main variants of the proportion method developed by the Institut Pasteur, Paris, and a method for calibrating sensitivity tests.

Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and is the first membrane-bound shrimp Dscam to be reported.
Down syndrome cell adhesion molecule (Dscam) seems likely to play a key role in the "alternative adaptive immunity" that has been reported in invertebrates. Dscam consists of a cytoplasmic tail that is involved in signal transduction and a hypervariable extracellular region that might use a pathogen recognition mechanism similar to that used by the vertebrate antibodies. In our previous paper, we isolated a unique tail-less form of Dscam from Litopenaeus vannamei. In this study, we report the first membrane-bound form of shrimp Dscam: PmDscam was isolated from Penaeus monodon, and it occurred in both membrane-bound and tail-less forms. Phylogenetic analysis showed that while the crustacean Dscams from shrimp and water flea did not share a single subclade, they were distinct from the invertebrate Dscam-like molecules and from the insecta Dscams. In the extracellular region, the variable regions of PmDscam were located in N-terminal Ig2, N-terminal Ig3 and the entire Ig7 domain. The PmDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events. The cytoplasmic tail variants were produced by exon inclusion/exclusion. Based on the genomic organization of Daphnia Dscam's cytoplasmic tail, we propose a model of how the shrimp Dscam genomic locus might use Type III polyadenylation to generate both the tail-less and membrane-bound forms.

[Pharmacokinetics and biotransformation of the antimycotic drug ciclopiroxolamine in animals and man after topical and systemic administration].
1. Following the dermal application of the carbon-14 labelled broad spectrum antimycotic 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone, 2-aminoethanol salt (ciclopiroxolamine, Hoe 296, Batrafen) in the form of a 1% aqueous cream to healthy human dorsal skin (penetration time: 6 h; occlusive dressing for 5 h), percutaneous absorption accounted on average for 1.3% of the dose applied. Excretion occurred via the kidney, with biological half-lives of 1.7 h. As can be seen from penetration studies of cadaverous skin, the horny layer contained the highest concentrations, with values of 2300-4500 microgram/cm3. The levels determined in the corium were still above the minimum inhibitory concentrations. These concentrations were already obtained at the first test stage (1.5 h after application) and did not change virtually at all over the longer penetration period. According to studies using histoautoradiography, ciclopirox can penetrate the skin via the epidermis and the hair follicles. When ciclopirox-14C-olamine aqueous cream was spread on the surface of fingernails, the radioactive-labelled compound penetrated right through the nail. The percutaneous absorption in dogs was higher, at 5-15% of the dose, than it was in humans. 2. After vaginal application (1 mg/kg) of ciclopirox-14C-olamine in the form of a 1% aqueous cream to bitches, between 42 and 97% of the dose (depending on the animal) was recovered in the urine and faeces, the remainder having penetrated into the tampon used to close the vagina. 3. Ciclopirox is excreted by dogs and man in the urine, primarily as a glucuronide. In humans another glucuronide with properties similar to those of the original substance was detected. Two conjugated, relatively non-polar metabolites were also present in small amounts. The metabolite patterns after oral and dermal application were similar. The binding of ciclopirox to serum proteins in humans was 96 +/- 2% in a concentration range of 0.01-11.0 microgram/ml. 4. Placental transfer was low in the rats studied. Though there was good absorption by the mother animal, the radioactivity in the foetal tissues was always lower than that of the maternal blood.

Development of a syngeneic mouse model for events related to ovarian cancer.
Mouse ovarian surface epithelial cells (MOSEC) were obtained from virgin, mature mice by mild trypsinization and were repeatedly passaged in vitro. Early passage cells (<20 passages) exhibited a cobblestone morphology and contact inhibition of growth. After approximately 20 passages in vitro, cobblestone morphology and contact inhibition of growth was lost. Tumor forming potential was determined by s.c. and i.p. injection of early and late passage cells into athymic and syngeneic C57BL6 mice. Subcutaneous tumors formed in approximately 4 months and were present only at the injection site. Intraperitoneal injection of late passage MOSEC into athymic and syngeneic mice resulted in growth of tumor implants throughout the abdominal cavity, and production of hemorrhagic ascitic fluid. Early passage MOSEC did not form tumors in vivo. Histopathologic analysis of tumors revealed a highly malignant neoplasm containing both carcinomatous and sarcomatous components. Late passage MOSEC expressed cytokeratin and did not produce ovarian steroids in response to gonadotropin stimulation in vitro. Ten clonal lines were established from late passage MOSEC. Each clone formed multiple peritoneal tumors and ascitic fluid after i.p. injection into C57BL6 mice. Three cell lines examined cytogenetically were polyploid with near-tetraploid modal chromosome numbers. Common clonal chromosome gains and losses included +5, +15, +19 and -X, -3, -4. One cell line had a clonal translocation between chromosomes 15 and 18 and another had a small marker chromosome; common structural abnormalities were not observed. These data describe the development of a mouse model for the study of events related to ovarian cancer in humans. The ability of the MOSEC to form extensive tumors within the peritoneal cavity, similar to those seen in women with Stage III and IV cancer, and the ability of the MOSEC to produce tumors in mice with intact immune systems, makes this model unique for investigations of molecular and immune interactions in ovarian cancer development.

Psychiatric comorbidity in chronic daily headache.
Clinical evidence suggests that chronic daily headache (CDH) occurs in association with psychopathologies: previous studies have focused particularly on migraine. To evaluate this association, we studied, using the DSM-IIIR criteria, a population of 88 patients (18M, 70F) affected by CDH (mean duration 7.4 +/- 8.7 years). We documented the presence of a psychiatric disorder in 90% of this population. The most frequent diagnosis was a comorbidity of anxiety and mood disorders. The comorbidity of psychiatric disorders and headache has important implications as far as treatment is concerned.

The use of mouse models to understand and improve cognitive deficits in Down syndrome
Remarkable advances have been made in recent years towards therapeutics for cognitive impairment in individuals with Down syndrome (DS) by using mouse models. In this review, we briefly describe the phenotypes of mouse models that represent outcome targets for drug testing, the behavioral tests used to assess impairments in cognition and the known mechanisms of action of several drugs that are being used in preclinical studies or are likely to be tested in clinical trials. Overlaps in the distribution of targets and in the pathways that are affected by these diverse drugs in the trisomic brain suggest new avenues for DS research and drug development.

Instruction: Given a scientific claim, retrieve documents that support or refute the claim


Question: Energy balance requires hypothalamic glutamate neurotransmission.

Synaptic glutamate release by ventromedial hypothalamic neurons is part of the neurocircuitry that prevents hypoglycemia.
The importance of neuropeptides in the hypothalamus has been experimentally established. Due to difficulties in assessing function in vivo, the roles of the fast-acting neurotransmitters glutamate and GABA are largely unknown. Synaptic vesicular transporters (VGLUTs for glutamate and VGAT for GABA) are required for vesicular uptake and, consequently, synaptic release of neurotransmitters. Ventromedial hypothalamic (VMH) neurons are predominantly glutamatergic and express VGLUT2. To evaluate the role of glutamate release from VMH neurons, we generated mice lacking VGLUT2 selectively in SF1 neurons (a major subset of VMH neurons). These mice have hypoglycemia during fasting secondary to impaired fasting-induced increases in the glucose-raising pancreatic hormone glucagon and impaired induction in liver of mRNAs encoding PGC-1alpha and the gluconeogenic enzymes PEPCK and G6Pase. Similarly, these mice have defective counterregulatory responses to insulin-induced hypoglycemia and 2-deoxyglucose (an antimetabolite). Thus, glutamate release from VMH neurons is an important component of the neurocircuitry that functions to prevent hypoglycemia.

Endometrial carcinoma risk among women diagnosed with endometrial hyperplasia: the 34-year experience in a large health plan
Classifying endometrial hyperplasia (EH) according to the severity of glandular crowding (simple hyperplasia (SH) vs complex hyperplasia (CH)) and nuclear atypia (simple atypical hyperplasia (SAH) vs complex atypical hyperplasia (CAH)) should predict subsequent endometrial carcinoma risk, but data on progression are lacking. Our nested case–control study of EH progression included 138 cases, who were diagnosed with EH and then with carcinoma (1970–2003) at least 1 year (median, 6.5 years) later, and 241 controls, who were individually matched on age, date, and follow-up duration and counter-matched on EH classification. After centralised pathology panel and medical record review, we generated rate ratios (RRs) and 95% confidence intervals (CIs), adjusted for treatment and repeat biopsies. With disordered proliferative endometrium (DPEM) as the referent, AH significantly increased carcinoma risk (RR=14, 95% CI, 5–38). Risk was highest 1–5 years after AH (RR=48, 95% CI, 8–294), but remained elevated 5 or more years after AH (RR=3.5, 95% CI, 1.0–9.6). Progression risks for SH (RR=2.0, 95% CI, 0.9–4.5) and CH (RR=2.8, 95% CI, 1.0–7.9) were substantially lower and only slightly higher than the progression risk for DPEM. The higher progression risks for AH could foster management guidelines based on markedly different progression risks for atypical vs non-atypical EH.

Management of chronic tension-type headache with tricyclic antidepressant medication, stress management therapy, and their combination: a randomized controlled trial.
CONTEXT Chronic tension-type headaches are characterized by near-daily headaches and often are difficult to manage in primary practice. Behavioral and pharmacological therapies each appear modestly effective, but data are lacking on their separate and combined effects. OBJECTIVE To evaluate the clinical efficacy of behavioral and pharmacological therapies, singly and combined, for chronic tension-type headaches. DESIGN AND SETTING Randomized placebo-controlled trial conducted from August 1995 to January 1998 at 2 outpatient sites in Ohio. PARTICIPANTS Two hundred three adults (mean age, 37 years; 76% women) with diagnosis of chronic tension-type headaches (mean, 26 headache d/mo). INTERVENTIONS Participants were randomly assigned to receive tricyclic antidepressant (amitriptyline hydrochloride, up to 100 mg/d, or nortriptyline hydrochloride, up to 75 mg/d) medication (n = 53), placebo (n = 48), stress management (eg, relaxation, cognitive coping) therapy (3 sessions and 2 telephone contacts) plus placebo (n = 49), or stress management therapy plus antidepressant medication (n = 53). MAIN OUTCOME MEASURES Monthly headache index scores calculated as the mean of pain ratings (0-10 scale) recorded by participants in a daily diary 4 times per day; number of days per month with at least moderate pain (pain rating >/=5), analgesic medication use, and Headache Disability Inventory scores, compared by intervention group. RESULTS Tricyclic antidepressant medication and stress management therapy each produced larger reductions in headache activity, analgesic medication use, and headache-related disability than placebo, but antidepressant medication yielded more rapid improvements in headache activity. Combined therapy was more likely to produce clinically significant (>/=50%) reductions in headache index scores (64% of participants) than antidepressant medication (38% of participants; P =.006), stress management therapy (35%; P =.003), or placebo (29%; P =.001). On other measures the combined therapy and its 2 component therapies produced similar outcomes. CONCLUSIONS Our results indicate that antidepressant medication and stress management therapy are each modestly effective in treating chronic tension-type headaches. Combined therapy may improve outcome relative to monotherapy.

Selective inhibition of Ras-dependent cell growth by farnesylthiosalisylic acid.
S-trans,trans-Farnesylthiosalicylic acid (FTS) is a novel farnesylated rigid carboxylic acid derivative. In cell-free systems, it acts as a potent competitive inhibitor (Ki = 2.6 microM) of the enzyme prenylated protein methyltransferase (PPMTase), which methylates the carboxyl-terminal S-prenylcysteine in a large number of prenylated proteins including Ras. In such systems, FTS inhibits Ras methylation but not Ras farnesylation. Inhibition of the PPMTase by FTS in homogenates or membranes of a variety of tissues and cell lines is inferred from a block in the methylation of exogenously added substrates such as N-acetyl-S-trans,trans-farnesyl-L-cysteine and of endogenous substrates including small GTP-binding proteins. FTS can also inhibit methylation of these proteins in intact cells (e.g. in Rat-1 fibroblasts, Ras-transformed Rat-1, and B16 melanoma cells). Unlike in cell-free systems, however, relatively high concentrations of FTS (50-100 microM) are required for partial blocking (10-40%) of protein methylation in the intact cells. Thus, FTS is a weak inhibitor of methylation in intact cells. Because methylation is the last step in the processing of Ras and related proteins, FTS is not likely to affect steps that precede it, e.g. protein prenylation. This may explain why the growth and gross morphology of a variety of cultured cell types (including Chinese hamster ovary, NIH3T3, Rat1, B16 melanoma, and PC12) is not affected by up to 25 microM FTS and is consistent with the observed lack of FTS-induced cytotoxicity. Nevertheless, FTS reduces the levels of Ras in cell membranes and can inhibit Ras-dependent cell growth in vitro, independently of methylation. It inhibits the growth of human Ha-ras-transformed cells (EJ cells) and reverses their transformed morphology in a dose-dependent manner (0.1-10 microM). The drug does not interfere with the growth of cells transformed by v-Raf or T-antigen but inhibits the growth of ErbB2-transformed cells and blocks the mitogenic effects of epidermal and basic fibroblast growth factors, thus implying its selectivity toward Ras growth signaling, possibly via modulation of Ras-Raf communication. Taken together, the results raise the possibility that FTS may specifically interfere with the interaction of Ras with a farnesylcysteine recognition domain in the cell membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Central obesity and hyperinsulinaemia in women are associated with polymorphism in the 5' flanking region of the human insulin gene.
Obesity is a multifactorial disease with a marked genetic component. The situation is further complicated by the heterogeneity of obesity demonstrated by the topographical distribution of body fat, e.g. upper body (central) and lower body (gluteal) obesity. Furthermore, the distribution of fat shows a stronger heritable tendency compared with total body fat. Central obesity is characterized by hyperinsulinaemia and insulin resistance, a feature in common with non-insulin dependent diabetes mellitus, hypertension and atherosclerosis. In order to study the molecular genetics of central obesity we have examined 56 severely obese (mean body mass index 40), unrelated British Caucasoid young non-diabetic women for associations of restriction fragment length polymorphism of candidate genes with anthropometric measurements and indices of insulin secretion and resistance. The candidate genes examined were insulin receptor, insulin sensitive glucose transporter and insulin. An association of the class 3 allele of the hypervariable region in the 5' flanking region of the insulin gene was found with upper segment obesity (P = 0.005). Furthermore, the class 3 allele was also associated with fasting hyperinsulinaemia (P = 0.01), stimulated insulin secretion (P = 0.01) and insulin resistance as calculated from the homeostatic model of assessment (HOMA; P = 0.008). No such associations were found with the other candidate genes studied. This data suggests that polymorphisms in the 5' flanking region of the insulin gene may affect expression of the gene and thereby modulate insulin production in severely obese female subjects.

Vitamin D status: measurement, interpretation, and clinical application.
Vitamin D, the sunshine vitamin, is now recognized not only for its importance in promoting bone health in children and adults but also for other health benefits, including reducing the risk of chronic diseases such as autoimmune diseases, common cancer, and cardiovascular disease. Vitamin D made in the skin or ingested in the diet is biologically inert and requires 2 successive hydroxylations first in the liver on carbon 25 to form 25-hydroxyvitamin D [25(OH)D], and then in the kidney for a hydroxylation on carbon 1 to form the biologically active form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)(2)D]. With the identification of 25(OH)D and 1,25(OH)(2)D, methods were developed to measure these metabolites in the circulation. Serum 25(OH)D is the barometer for vitamin D status. Serum 1,25(OH)(2)D provides no information about vitamin D status and is often normal or even increased as the result of secondary hyperparathyroidism associated with vitamin D deficiency. Most experts agree that 25(OH)D of <20 ng/mL is considered to be vitamin D deficiency, whereas a 25(OH)D of 21-29 ng/mL is considered to be insufficient. The goal should be to maintain both children and adults at a level >30 ng/mL to take full advantage of all the health benefits that vitamin D provides.

Transformation of mammalian cells by constitutively active MAP kinase kinase.
Mitogen-activated protein (MAP) kinase kinase (MAPKK) activates MAP kinase in a signal transduction pathway that mediates cellular responses to growth and differentiation factors. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells by prolonging the activated state of MAPKK and of components downstream in the signaling pathway. To test this hypothesis, constitutively active MAPKK mutants were designed that had basal activities up to 400 times greater than that of the unphosphorylated wild-type kinase. Expression of these mutants in mammalian cells activated AP-1-regulated transcription. The cells formed transformed foci, grew efficiently in soft agar, and were highly tumorigenic in nude mice. These findings indicate that constitutive activation of MAPKK is sufficient to promote cell transformation.

Atherosclerotic plaque rupture--pathologic basis of plaque stability and instability.
Time for primary reveiw 27 days   Atherosclerosis continues to be one of the main subjects in pathology research. The intriguing complexity of its pathogenesis as well as the importance of its clinical sequelae provide a rationale for this [1]. A large number of diseases with totally different clinical presentations are basically atherosclerosis related, and among these, myocardial infarction, stroke, abdominal aneurysms and lower limb ischemia determine to a large extent the morbidity and mortality in Western style populations. But, despite this broad spectrum of clinical disease, most of the acute manifestations of atherosclerosis share a common pathogenetic feature: rupture of an atherosclerotic plaque [2–4]. Plaque disruptions may vary greatly in extent from tiny fissures or erosions of the plaque surface to deep intimal tears which extend into the soft lipid core of lesions; in all these instances, at least some degree of thrombus formation occurs [5, 6]. The abdominal aorta is the arterial site most prominently involved in the process of plaque formation, and also of plaque complications. In this large diameter vessel the process of plaque disruption and thrombosis is not ended by luminal occlusion, and may lead to extensive surface ulcerations comprising large areas of the aortic wall, as can be observed in many autopsy cases at older age. Apart from the undisputable role of atherosclerosis in abdominal aneurysm formation [7], mural thrombosis leads to a surprisingly low rate of clinically significant complications in these patients, although cholesterol emboli can be regularly found in their kidneys and skin at autopsy. Still, it is presently unclear what impact the various biologically active mediators released from eroded aortic surfaces may have on the human body. In contrast, in small diameter vessels such as coronary arteries, occlusive thrombosis is a frequent and often fatal complication of plaque …   * Corresponding author. Tel.: +31-20-5665-633; fax: +31-20-914-738; e-mail a.c.vanderwal@amc.uva.nl

Ribosome biogenesis: Achilles heel of cancer?
It is long been known that cancer and non-cancer cells can be distinguished on the basis of their nucleolar morphologies. As early as the 19th century, it was reported that cancer cells have larger and more irregularly shaped nucleoli. Since then, pathologists have used nucleolar morphology to predict the clinical outcome [1]. Nucleolar morphology is altered due to the up-regulation of ribosomal gene transcription. Within nucleoli, ribosomal genes (rDNA) are transcribed by RNA polymerase I (pol I). The pre-ribosomal RNA (pre-rRNA) transcripts are subsequently modified and processed into the mature 18S, 5.8S and 28S rRNAs. 5S rRNA is transcribed by RNA polymerase III in the nucleoplasm. Together with the ribosomal proteins, the 5S rRNA is imported into the nucleolus where 40S and 60S ribosomal subunits are assembled prior to export to the cytoplasm [1, 2]. Oncogenes such as c-Myc can both directly and indirectly upregulate rDNA transcription, while tumour suppressors like p53 and Rb suppress ribosome biogenesis. Mutations in these genes not only result in deregulated cell cycle control, but also upregulated ribosome biogenesis. In addition to ribosome biogenesis, the nucleolus is a key cellular stress sensor and plays a central role in p53 activation [1, 2]. The increased translational capacity of cancer cells enables them to maintain higher proliferation rates. As stated by Ruggero, “compared with normal cells, cancer cells may be addicted to increases in ribosome biogenesis and number” [1]. This provides new therapeutic opportunities. As it turns out many chemotherapeutic drugs used in cancer treatment already inhibit ribosome biogenesis. In one recent survey it was shown that 20 out of 36 drugs in clinical use inhibit ribosome biogenesis [3]. Most of these drugs were originally designed to target highly proliferating cells by damaging DNA, interfering with DNA synthesis or with mitosis. These targeting modalities of these drugs also lead to toxicity in normal highly proliferating tissues. An example is ActinomycinD (AMD), a DNA intercalator which has a preference for GC-rich DNA sequences. As rDNA has above average GC-richness and because of its open chromatin conformation, low concentrations of AMD preferentially inhibit RNA polymerase I transcription and upon prolonged exposure causes genome wide DNA damage. Alkylating drugs like cisplatin and oxaliplatin or topoisomerases poisons like camptothecin inhibit pol I transcription. The degree to which inhibition of ribosome biogenesis contributes to the efficacy of these drugs is difficult to establish [3]. This raises an important question. Can targeting ribosome biogenesis without DNA damage offer any therapeutic potential? Two recently described drugs CX-5461 and BMH-21 are now providing evidence that inhibition of ribosome biogenesis by targeting transcription of rDNA by pol I has promising therapeutic potential. CX-5461 was designed to specifically inhibit pol I transcription by disrupting pre-initiation complex formation at the rDNA promoter. CX-5461 has been shown to activate p53 via nucleolar stress. It induces autophagy as well as senescence in a multiple types of cancer cells in a p53-dependent manner. Especially in leukaemia and lymphoma cells, treatment with CX-5461 induces p53-dependent apoptosis, while normal cells tolerate it [4, 5]. Whether the drug also induces DNA damage was not fully addressed, but it was demonstrated that it could induce cell death in cells lacking ATM - a key mediator of DNA double strand break responses. However, more recently Laiho and colleagues have shown that at high concentrations, CX-5461 does induce a γH2AX response, raising concerns about DNA damage [6]. BMH-21 was identified in a screen performed by Laiho and colleagues aimed at identifying novel p53 activators. Like AMD, BMH-21 is a DNA intercalator with preference for GC rich sequences [7]. Continuing the parallel with AMD, BMH-21 is a potent and specific inhibitor rDNA transcription and induces nucleolar reorganisation often referred to as nucleolar capping. Interestingly, transcription inhibition was followed by the degradation of the main pol I subunit, RPA194, by the proteasome [6]. In contrast with AMD, initial indications were that BMH-21 did not appear to induce DNA damage as evidenced by the lack of a γH2AX response [7]. Inhibition of transcription by BMH-21 causes nucleolar stress, resulting in decreased proliferation and cell death. P53 is activated in BMH-21 treated cells but is not required for its anti-proliferative effects. Intriguingly, it appears that cancer cells with high demands for ribosome biogenesis are selectively targeted [6]. The current publication in Oncotarget now rules out any role for DNA damage signalling and repair pathways in the BMH-21 response. Moreover, BMH-21 derivatives that can induce DNA damage display lower efficiency in inducing nucleolar stress and inhibiting proliferation [8]. The central importance of this study is that it finally uncouples DNA damage and nucleolar stress and reveals an Achilles heel in cancer cells, their addiction to ribosome biogenesis.

Peroxisome proliferator-activated receptor ligand bezafibrate for prevention of type 2 diabetes mellitus in patients with coronary artery disease.
BACKGROUND Recent studies have shown that type 2 diabetes is preventable by both lifestyle interventions and medications that influence primary glucose metabolism. Whether pharmacological interventions that influence primary lipid metabolism can also delay development of type 2 diabetes is unknown. The goal of this study was to evaluate the effect of the peroxisome proliferator-activated receptor ligand bezafibrate on the progression of impaired fasting glucose phase to type 2 diabetes in patients with coronary artery disease over a 6.2-year follow-up period. METHODS AND RESULTS The study sample comprised 303 nondiabetic patients 42 to 74 years of age with a fasting blood glucose level of 110 to 125 mg/dL (6.1 to 6.9 mmol/L). The patients received either 400 mg bezafibrate retard (156 patients) or placebo (147 patients) once a day. No patients were using statins, and use of ACE inhibitors, which also reduce diabetes incidence, was relatively low. During follow-up, development of new-onset diabetes was recorded in 146 patients: in 80 (54.4%) from the placebo group and 66 (42.3%) from the bezafibrate group (P=0.04). The mean time until onset of new diabetes was significantly delayed in patients on bezafibrate compared with patients on placebo: 4.6+/-2.3 versus 3.8+/-2.6 years (P=0.004). Multivariate analysis identified bezafibrate treatment as an independent predictor of reduced risk of new diabetes development (hazard ratio, 0.70; 95% CI, 0.49 to 0.99). Other significant variables associated with future overt type 2 diabetes in patients with impaired fasting glucose were total cholesterol level (hazard ratio, 1.22; 95% CI 1.0 to 1.51) and body mass index (hazard ratio, 1.10; 95% CI, 1.05 to 1.16). CONCLUSIONS Bezafibrate reduces the incidence and delays the onset of type 2 diabetes in patients with impaired fasting glucose. Whether the combination of bezafibrate with other recommended drugs for secondary prevention (statins and ACE inhibitors) would be as efficacious as suggested by our results remains to be determined.

Acute and chronic exercise in patients with heart failure with reduced ejection fraction: evidence of structural and functional plasticity and intact angiogenic signalling in skeletal muscle
KEY POINTS The vascular endothelial growth factor (VEGF) responses to acute submaximal exercise and training effects in patients with heart failure with reduced ejection fraction (HFrEF) were investigated. Six patients and six healthy matched controls performed knee-extensor exercise (KE) at 50% of maximum work rate before and after (only patients) KE training. Muscle biopsies were taken to assess skeletal muscle structure and the angiogenic response. Before training, during this submaximal KE exercise, patients with HFrEF exhibited higher leg vascular resistance and greater noradrenaline spillover. Skeletal muscle structure and VEGF response were generally not different between groups. Following training, resistance was no longer elevated and noradrenaline spillover was curtailed in the patients. Although, in the trained state, VEGF did not respond to acute exercise, capillarity was augmented. Muscle fibre cross-sectional area and percentage area of type I fibres increased and mitochondrial volume density exceeded that of controls. Structural/functional plasticity and appropriate angiogenic signalling were observed in skeletal muscle of patients with HFrEF. ABSTRACT This study examined the response to acute submaximal exercise and the effect of training in patients with heart failure with reduced ejection fraction (HFrEF). The acute angiogenic response to submaximal exercise in HFrEF after small muscle mass training is debated. The direct Fick method, with vascular pressures, was performed across the leg during knee-extensor exercise (KE) at 50% of maximum work rate (WRmax ) in patients (n = 6) and controls (n = 6) and then after KE training in patients. Muscle biopsies facilitated the assessment of skeletal muscle structure and vascular endothelial growth factor (VEGF) mRNA levels. Prior to training, HFrEF exhibited significantly higher leg vascular resistance (LVR) (≈15%) and significantly greater noradrenaline spillover (≈385%). Apart from mitochondrial volume density, which was significantly lower (≈22%) in HFrEF, initial skeletal muscle structure, including capillarity, was not different between groups. Resting VEGF mRNA levels, and the increase with exercise, was not different between patients and controls. Following training, LVR was no longer elevated and noradrenaline spillover was curtailed. Skeletal muscle capillarity increased with training, as assessed by capillary-to-fibre ratio (≈13%) and number of capillaries around a fibre (NCAF ) (≈19%). VEGF mRNA was now not significantly increased by acute exercise. Muscle fibre cross-sectional area and percentage area of type I fibres both increased significantly with training (≈18% and ≈21%, respectively), while the percentage area of type II fibres fell significantly (≈11%), and mitochondrial volume density now exceeded that of controls. These data reveal structural and functional plasticity and appropriate angiogenic signalling in skeletal muscle of HFrEF patients.

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Question: Energy balance requires hypothalamic glutamate neurotransmission.

Topographic mapping of VMH → arcuate nucleus microcircuits and their reorganization by fasting
In the hypothalamic arcuate nucleus (ARC), pro-opiomelanocortin (POMC) neurons inhibit feeding and neuropeptide-Y (NPY) neurons stimulate feeding. We tested whether neurons in the ventromedial hypothalamic nucleus (VMH), a known satiety center, activate anorexigenic neuronal pathways in the ARC by projecting either excitatory synaptic inputs to POMC neurons and/or inhibitory inputs to NPY neurons. Using laser scanning photostimulation in brain slices from transgenic mice, we found that POMC and NPY neurons, which are interspersed in the ARC, are nevertheless regulated by anatomically distinct synaptic inputs. POMC neurons received strong excitatory input from the medial VMH (mVMH), whereas NPY neurons did not and, instead, received weak inhibitory input only from within the ARC. The strength of the excitatory input from the mVMH to POMC neurons was diminished by fasting. These data identify a new molecularly defined circuit that is dynamically regulated by nutritional state in a manner consistent with the known role of the VMH as a satiety center.

Population density profiles of nasopharyngeal carriage of 5 bacterial species in pre-school children measured using quantitative PCR offer potential insights into the dynamics of transmission.
Bacterial vaccines can reduce carriage rates. Colonization is usually a binary endpoint. Real time quantitative PCR (qPCR) can quantify bacterial DNA in mucosal samples over a wide range. Using culture and single-gene species-specific qPCRs for Streptococcus pneumoniae (lytA), Streptococcus pyogenes (ntpC), Moraxella catarrhalis (ompJ), Haemophilus influenzae (hdp) and Staphylococcus aureus (nuc) and standard curves against log-phase reference strain broth cultures we described frequency and peak density distributions of carriage in nasopharyngeal swabs from 161 healthy 2-4 y old children collected into STGG broth. In general, detection by qPCR and culture was consistent. Discordance mostly occurred at lower detection thresholds of both methods, although PCR assays for S. pyogenes and S. aureus were less sensitive. Density varied across 5-7 orders of magnitude for the 5 species with the abundant species skewed toward high values (modes: S. pneumoniae log3-4, M. catarrhalis & H. influenzae log4-5 CFU/ml broth). Wide ranges of bacterial DNA concentrations in healthy children carrying these bacteria could mean that different individuals at different times vary greatly in infectiousness. Understanding the host, microbial and environmental determinants of colonization density will permit more accurate prediction of vaccine effectiveness.

PRRSV receptors and their roles in virus infection
Porcine reproductive and respiratory syndrome virus (PRRSV) has a restricted cell tropism and prefers to invade well-differentiated cells of the monocyte/macrophage lineage, such as pulmonary alveolar macrophages and African green monkey kidney cell line MA-104 and its derivatives, such as Marc-145, Vero and CL-2621. PRRSV infection of the host cells actually is a receptor-mediated endocytosis and replication process. The presence and absence of the cellular receptors decide whether the cell lines are permissive or non-permissive to PRRSV infection. Several PRRSV non-permissive cell lines, such as BHK-21, PK-15 and CHO-K1, have been shown to become sensitive to the virus infection upon expression of the recombinant receptor proteins. Up to now, heparin sulfate, sialoadhesin, CD163, CD151 and vimentin have been identified as the important PRRSV receptors via their involvement in virus attachment, internalization or uncoating. Each receptor is characterized by the distribution in different cells, the function in virus different infection stages and the interaction model with the viral proteins or genes. Joint forces of the receptors recently attract attentions due to the specific function. PRRSV receptors have become the targets for designing the new anti-viral reagents or the recombinant cell lines used for isolating the viruses or developing more effective vaccines due to their more conserved sequences compared with the genetic variation of the virus. In this paper, the role of PRRSV receptors and the molecular mechanism of the interaction between the virus and the receptors are reviewed.

The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3).
IMPORTANCE Definitions of sepsis and septic shock were last revised in 2001. Considerable advances have since been made into the pathobiology (changes in organ function, morphology, cell biology, biochemistry, immunology, and circulation), management, and epidemiology of sepsis, suggesting the need for reexamination. OBJECTIVE To evaluate and, as needed, update definitions for sepsis and septic shock. PROCESS A task force (n = 19) with expertise in sepsis pathobiology, clinical trials, and epidemiology was convened by the Society of Critical Care Medicine and the European Society of Intensive Care Medicine. Definitions and clinical criteria were generated through meetings, Delphi processes, analysis of electronic health record databases, and voting, followed by circulation to international professional societies, requesting peer review and endorsement (by 31 societies listed in the Acknowledgment). KEY FINDINGS FROM EVIDENCE SYNTHESIS Limitations of previous definitions included an excessive focus on inflammation, the misleading model that sepsis follows a continuum through severe sepsis to shock, and inadequate specificity and sensitivity of the systemic inflammatory response syndrome (SIRS) criteria. Multiple definitions and terminologies are currently in use for sepsis, septic shock, and organ dysfunction, leading to discrepancies in reported incidence and observed mortality. The task force concluded the term severe sepsis was redundant. RECOMMENDATIONS Sepsis should be defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. For clinical operationalization, organ dysfunction can be represented by an increase in the Sequential [Sepsis-related] Organ Failure Assessment (SOFA) score of 2 points or more, which is associated with an in-hospital mortality greater than 10%. Septic shock should be defined as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone. Patients with septic shock can be clinically identified by a vasopressor requirement to maintain a mean arterial pressure of 65 mm Hg or greater and serum lactate level greater than 2 mmol/L (>18 mg/dL) in the absence of hypovolemia. This combination is associated with hospital mortality rates greater than 40%. In out-of-hospital, emergency department, or general hospital ward settings, adult patients with suspected infection can be rapidly identified as being more likely to have poor outcomes typical of sepsis if they have at least 2 of the following clinical criteria that together constitute a new bedside clinical score termed quickSOFA (qSOFA): respiratory rate of 22/min or greater, altered mentation, or systolic blood pressure of 100 mm Hg or less. CONCLUSIONS AND RELEVANCE These updated definitions and clinical criteria should replace previous definitions, offer greater consistency for epidemiologic studies and clinical trials, and facilitate earlier recognition and more timely management of patients with sepsis or at risk of developing sepsis.

CD34+ hematopoietic stem-progenitor cell microRNA expression and function: a circuit diagram of differentiation control.
MicroRNAs (miRNAs) are a recently identified class of epigenetic elements consisting of small noncoding RNAs that bind to the 3' untranslated region of mRNAs and down-regulate their translation to protein. miRNAs play critical roles in many different cellular processes including metabolism, apoptosis, differentiation, and development. We found 33 miRNAs expressed in CD34+ hematopoietic stem-progenitor cells (HSPCs) from normal human bone marrow and mobilized human peripheral blood stem cell harvests. We then combined these data with human HSPC mRNA expression data and with miRNA-mRNA target predictions, into a previously undescribed miRNA:mRNA interaction database called the Transcriptome Interaction Database. The in silico predictions from the Transcriptome Interaction Database pointed to miRNA control of hematopoietic differentiation through translational control of mRNAs critical to hematopoiesis. From these predictions, we formulated a model for miRNA control of stages of hematopoiesis in which many of the genes specifying hematopoietic differentiation are expressed by HSPCs, but are held in check by miRNAs until differentiation occurs. We validated miRNA control of several of these target mRNAs by demonstrating that their translation in fact is decreased by miRNAs. Finally, we chose miRNA-155 for functional characterization in hematopoiesis, because we predicted that it would control both myelopoiesis and erythropoiesis. As predicted, miRNA-155 transduction greatly reduced both myeloid and erythroid colony formation of normal human HSPCs.

Overview of the CCP4 suite and current developments
The CCP4 (Collaborative Computational Project, Number 4) software suite is a collection of programs and associated data and software libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims. The programs are from a wide variety of sources but are connected by a common infrastructure provided by standard file formats, data objects and graphical interfaces. Structure solution by macromolecular crystallography is becoming increasingly automated and the CCP4 suite includes several automation pipelines. After giving a brief description of the evolution of CCP4 over the last 30 years, an overview of the current suite is given. While detailed descriptions are given in the accompanying articles, here it is shown how the individual programs contribute to a complete software package.

Role of inscuteable in orienting asymmetric cell divisions in Drosophila
Drosophila neuroblasts and epithelial cells in the procephalic neurogenic region divide perpendicular to the surface, and segregate the proteins Numb and Prospero into the basal daughter cell. We demonstrate here that orientation of the mitotic spindle and correct localization of Numb and Prospero in these cells require the inscuteable gene. Moreover, ectopic expression of inscuteable in other epithelial cells leads to spindle reorientation. The Inscuteable protein localizes to the apical cell cortex before mitosis, suggesting that Inscuteable functions in establishing polarity for asymmetric cell division.

Receptor tyrosine kinases endocytosis in endothelium: biology and signaling.
Receptor tyrosine kinases are involved in regulation of key processes in endothelial biology, including proliferation, migration, and angiogenesis. It is now generally accepted that receptor tyrosine kinase signaling occurs intracellularly and on the plasma membrane, although many important details remain to be worked out. Endocytosis and subsequent intracellular trafficking spatiotemporally regulate receptor tyrosine kinase signaling, whereas signaling endosomes provide a platform for the compartmentalization of signaling events. This review summarizes recent advances in our understanding of endothelial receptor tyrosine kinase endocytosis and signaling using vascular endothelial growth factor receptor-2 as a paradigm.

Keratin-dependent regulation of Aire and gene expression in skin tumor keratinocytes
Expression of the intermediate filament protein keratin 17 (K17) is robustly upregulated in inflammatory skin diseases and in many tumors originating in stratified and pseudostratified epithelia. We report that autoimmune regulator (Aire), a transcriptional regulator, is inducibly expressed in human and mouse tumor keratinocytes in a K17-dependent manner and is required for timely onset of Gli2-induced skin tumorigenesis in mice. The induction of Aire mRNA in keratinocytes depends on a functional interaction between K17 and the heterogeneous nuclear ribonucleoprotein hnRNP K. Further, K17 colocalizes with Aire protein in the nucleus of tumor-prone keratinocytes, and each factor is bound to a specific promoter region featuring an NF-κB consensus sequence in a relevant subset of K17- and Aire-dependent proinflammatory genes. These findings provide radically new insight into keratin intermediate filament and Aire function, along with a molecular basis for the K17-dependent amplification of inflammatory and immune responses in diseased epithelia.

The long non-coding RNA H19-derived miR-675 modulates human gastric cancer cell proliferation by targeting tumor suppressor RUNX1.
The lncRNA H19 has been recently shown to be upregulated and play important roles in gastric cancer tumorigenesis. However, the precise molecular mechanism of H19 and its mature product miR-675 in the carcinogenesis of gastric cancer remains unclear. In this study, we found that miR-675 was positively expressed with H19 and was a pivotal mediator in H19-induced gastric cancer cell growth promotion. Subsequently, the tumor suppressor Runt Domain Transcription Factor1 (RUNX1) was confirmed to be a direct target of miR-675 using a luciferase reporter assay and Western blotting analyses. A series of rescue assays indicated that RUNX1 mediated H19/miR-67-induced gastric cancer cell phenotypic changes. Moreover, the inverse relationship between the expression of RUNX1 and H19/miR-675 was also revealed in gastric cancer tissues and gastric cancer cell lines. Taken together, our study demonstrated that the novel pathway H19/miR-675/RUNX1 regulates gastric cancer development and may serve as a potential target for gastric cancer therapy.

Regulation of autophagy by cytoplasmic p53
Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53−/− cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

Instruction: Given a scientific claim, retrieve documents that support or refute the claim