Th17 and regulatory T (Treg) cells play opposite roles in autoimmune diseases. However, the mechanisms underlying their proper migration to inflammatory tissues are unclear. In this study, we report that these two T cell subsets both express CCR6. CCR6 expression in Th17 cells is regulated by TGF-beta and requires two nuclear receptors, RORalpha and RORgamma. Th17 cells also express the CCR6 ligand CCL20, which is induced synergistically by TGF-beta and IL-6, which requires STAT3, RORgamma and IL-21. Th17 cells, by producing CCL20, promote migration of Th17 and Treg cells in vitro in a CCR6-dependent manner. Lack of CCR6 in Th17 cells reduces the severity of experimental autoimmune encephalomyelitis and Th17 and Treg recruitment into inflammatory tissues. Similarly, CCR6 on Treg cells is also important for their recruitment into inflammatory tissues. Our data indicate an important role of CCR6 in Treg and Th17 cell migration.


The effect of omega-3, omega-6 and omega-9 unsaturated fatty acids (UFAs) on receptor-mediated Ca2+ entry was investigated in a T-cell line (JURKAT) by using anti-CD3 antibodies (OKT3) to induce intracellular Ca2+ [( Ca2+]i) increase and Ca2+ influx. All the UFAs, as well as Ni2+ ions and 12-O-tetradecanoylphorbol 13-acetate, decreased the OKT3-induced sustained [Ca2+]i increase to basal levels. Although non-esterified fatty acids activate protein kinase C (PKC) [McPhail, Clayton & Snyderman (1984) Science 224, 622-624; Murakami, Chan & Routtenberg (1986) J. Biol. Chem. 261, 15424-15429], studies using H-7 and analysis of the PKC-dependent phosphorylation of 19 and 80 kDa marker substrates ruled out the involvement of PKC in UFA-induced inhibition of Ca2+ entry. Flow-cytometry analysis showed that UFAs do not interfere with antibody-receptor binding. BSA (0.2%, w/v) reversed the effect of UFAs after these fatty acids have decreased the OKT3-induced [Ca2+]i increase to basal levels. The relevance of these findings and possible mechanisms for inhibition by UFAs of receptor-mediated Ca2+ influx were discussed.


BACKGROUND Pregnant women with type 1 diabetes are a high-risk population who are recommended to strive for optimal glucose control, but neonatal outcomes attributed to maternal hyperglycaemia remain suboptimal. Our aim was to examine the effectiveness of continuous glucose monitoring (CGM) on maternal glucose control and obstetric and neonatal health outcomes. METHODS In this multicentre, open-label, randomised controlled trial, we recruited women aged 18-40 years with type 1 diabetes for a minimum of 12 months who were receiving intensive insulin therapy. Participants were pregnant (≤13 weeks and 6 days' gestation) or planning pregnancy from 31 hospitals in Canada, England, Scotland, Spain, Italy, Ireland, and the USA. We ran two trials in parallel for pregnant participants and for participants planning pregnancy. In both trials, participants were randomly assigned to either CGM in addition to capillary glucose monitoring or capillary glucose monitoring alone. Randomisation was stratified by insulin delivery (pump or injections) and baseline glycated haemoglobin (HbA1c). The primary outcome was change in HbA1c from randomisation to 34 weeks' gestation in pregnant women and to 24 weeks or conception in women planning pregnancy, and was assessed in all randomised participants with baseline assessments. Secondary outcomes included obstetric and neonatal health outcomes, assessed with all available data without imputation. This trial is registered with ClinicalTrials.gov, number NCT01788527. FINDINGS Between March 25, 2013, and March 22, 2016, we randomly assigned 325 women (215 pregnant, 110 planning pregnancy) to capillary glucose monitoring with CGM (108 pregnant and 53 planning pregnancy) or without (107 pregnant and 57 planning pregnancy). We found a small difference in HbA1c in pregnant women using CGM (mean difference -0·19%; 95% CI -0·34 to -0·03; p=0·0207). Pregnant CGM users spent more time in target (68% vs 61%; p=0·0034) and less time hyperglycaemic (27% vs 32%; p=0·0279) than did pregnant control participants, with comparable severe hypoglycaemia episodes (18 CGM and 21 control) and time spent hypoglycaemic (3% vs 4%; p=0·10). Neonatal health outcomes were significantly improved, with lower incidence of large for gestational age (odds ratio 0·51, 95% CI 0·28 to 0·90; p=0·0210), fewer neonatal intensive care admissions lasting more than 24 h (0·48; 0·26 to 0·86; p=0·0157), fewer incidences of neonatal hypoglycaemia (0·45; 0·22 to 0·89; p=0·0250), and 1-day shorter length of hospital stay (p=0·0091). We found no apparent benefit of CGM in women planning pregnancy. Adverse events occurred in 51 (48%) of CGM participants and 43 (40%) of control participants in the pregnancy trial, and in 12 (27%) of CGM participants and 21 (37%) of control participants in the planning pregnancy trial. Serious adverse events occurred in 13 (6%) participants in the pregnancy trial (eight [7%] CGM, five [5%] control) and in three (3%) participants in the planning pregnancy trial (two [4%] CGM and one [2%] control). The most common adverse events were skin reactions occurring in 49 (48%) of 103 CGM participants and eight (8%) of 104 control participants during pregnancy and in 23 (44%) of 52 CGM participants and five (9%) of 57 control participants in the planning pregnancy trial. The most common serious adverse events were gastrointestinal (nausea and vomiting in four participants during pregnancy and three participants planning pregnancy). INTERPRETATION Use of CGM during pregnancy in patients with type 1 diabetes is associated with improved neonatal outcomes, which are likely to be attributed to reduced exposure to maternal hyperglycaemia. CGM should be offered to all pregnant women with type 1 diabetes using intensive insulin therapy. This study is the first to indicate potential for improvements in non-glycaemic health outcomes from CGM use. FUNDING Juvenile Diabetes Research Foundation, Canadian Clinical Trials Network, and National Institute for Health Research.


Human group 1 ILCs consist of at least three phenotypically distinct subsets, including NK cells, CD127(+) ILC1, and intraepithelial CD103(+) ILC1. In inflamed intestinal tissues from Crohn's disease patients, numbers of CD127(+) ILC1 increased at the cost of ILC3. Here we found that differentiation of ILC3 to CD127(+) ILC1 is reversible in vitro and in vivo. CD127(+) ILC1 differentiated to ILC3 in the presence of interleukin-2 (IL-2), IL-23, and IL-1β dependent on the transcription factor RORγt, and this process was enhanced in the presence of retinoic acid. Furthermore, we observed in resection specimen from Crohn's disease patients a higher proportion of CD14(+) dendritic cells (DC), which in vitro promoted polarization from ILC3 to CD127(+) ILC1. In contrast, CD14(-) DCs promoted differentiation from CD127(+) ILC1 toward ILC3. These observations suggest that environmental cues determine the composition, function, and phenotype of CD127(+) ILC1 and ILC3 in the gut.


Forkhead box transcription factor, class O (FOXO) is a mammalian homologue of DAF-16, which is known to regulate the lifespan of Caenorhabditis elegans and includes subfamilies of forkhead transcription factors such as AFX, FKHRL1, and FKHR. FKHR is phosphorylated on three sites (Thr-24, Ser-256, and Ser-319) in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner, thereby inhibiting death signals. We here documented dephosphorylation of FKHR following transient forebrain ischemia with its concomitant translocation into the nucleus in neurons in gerbil and mouse brains. The activation of FKHR preceded delayed neuronal death in the vulnerable hippocampal regions following ischemic brain injury. The FKHR activation was accompanied by an increase in DNA binding activity for FKHR-responsive element on the Fas ligand promoter. We also defined FKHR-induced downstream targets such as Fas ligand and Bim in brain ischemia. Therefore, we propose a new strategy to rescue neurons from delayed neuronal death by promoting the survival signaling. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, up-regulated Akt activity in the brain and in turn rescue neurons from delayed neuronal death by inhibiting FKHR-dependent or -independent death signals in neurons.


OBJECTIVE To describe the sources of protein intake in a sample of the US adult population and among subgroups defined by race-ethnicity, age, and gender. DESIGN The Third National Health and Nutrition Examination Survey, 1988-1991, is a stratified random sample of the total civilian noninstitutionalized population, drawn from the 50 United States and the District of Columbia. For all foods consumed by the participants, based on a 24-hour dietary recall, protein sources and the contribution of each protein type to the total protein intake were determined. SUBJECTS Adult participants in the third National Health and Nutrition Examination Survey (n = 7,924). STATISTICAL ANALYSES Weighted total, age-specific, and age-adjusted mean protein intakes were calculated using SAS and WesVarPC. Statistical differences were determined by 2-tailed t tests. RESULTS The main protein source in the American diet is animal protein (69%). Meat, fish, and poultry protein combined contributed the most to animal protein (42%), followed by dairy protein (20%). Grains (18%) contributed the most to plant protein consumption. Women consumed a lower percentage of beef (14%) and pork (7%) protein than did men (18% and 9%, respectively). Women also consumed a higher percentage of poultry (13%), dairy (22%), and fruit and vegetable (11%) protein than did men (11%, 19%, and 9%, respectively). Blacks reported eating a higher percentage of poultry (18%) and pork (11%) protein and a lower percent of dairy protein (14%) than did whites (12%, 7%, and 22%, respectively) and Mexican-Americans (11%, 8%, and 17%, respectively). Mexican-Americans consumed a higher percentage of legume (7%) and egg (7%) protein than did whites (4% and 4%, respectively) and blacks (4% and 5%, respectively). Whites consumed a higher percentage of grain protein (19%) than did blacks (16%) and Mexican-Americans (15%). CONCLUSIONS These results show that, although the percentage of total energy from protein may be similar among race-ethnicities and between men and women, their sources of protein are different. These differences should be taken into account when providing nutrition education for specific populations.


Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.


Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The role of altered sialylation in multiple myeloma (MM) cell trafficking has not been previously investigated. In the present study we identified high expression of β-galactoside α-2,3-sialyltransferase, ST3GAL6, in MM cell lines and patients. This gene plays a key role in selectin ligand synthesis in humans through the generation of functional sialyl Lewis X. In MRC IX patients, high expression of this gene is associated with inferior overall survival. In this study we demonstrate that knockdown of ST3GAL6 results in a significant reduction in levels of α-2,3-linked sialic acid on the surface of MM cells with an associated significant reduction in adhesion to MM bone marrow stromal cells and fibronectin along with reduced transendothelial migration in vitro. In support of our in vitro findings, we demonstrate significantly reduced homing and engraftment of ST3GAL6 knockdown MM cells to the bone marrow niche in vivo, along with decreased tumor burden and prolonged survival. This study points to the importance of altered glycosylation, particularly sialylation, in MM cell adhesion and migration.


There have been numerous epidemiologic investigations to determine factors that may affect cancer risk. There is also a rich tradition of evaluating potential somatic changes in the cancer itself to predict recurrence and/or mortality after diagnosis. However, there has been relatively little


Membrane cholesterol modulates a variety of cell signaling pathways and functions. While cholesterol depletion by high-density lipoproteins (HDLs) has potent anti-inflammatory effects in various cell types, its effects on inflammatory responses in macrophages remain elusive. Here we show overt pro-inflammatory effects of HDL-mediated passive cholesterol depletion and lipid raft disruption in murine and human primary macrophages in vitro. These pro-inflammatory effects were confirmed in vivo in peritoneal macrophages from apoA-I transgenic mice, which have elevated HDL levels. In line with these findings, the innate immune responses required for clearance of P. aeruginosa bacterial infection in lung were compromised in mice with low HDL levels. Expression analysis, ChIP-PCR, and combinatorial pharmacological and genetic intervention studies unveiled that both native and reconstituted HDL enhance Toll-like-receptor-induced signaling by activating a PKC-NF-κB/STAT1-IRF1 axis, leading to increased inflammatory cytokine expression. HDL's pro-inflammatory activity supports proper functioning of macrophage immune responses.


BACKGROUND A major challenge for physicians is to identify patients with acute coronary syndromes who may benefit from treatment with glycoprotein-IIb/IIIa-receptor antagonists. We investigated whether troponin concentrations can be used to stratify patients for benefit from treatment with tirofiban. METHODS We enrolled 2222 patients of the Platelet Receptor Inhibition in Ischemic Syndrome Management study with coronary artery disease and who had had chest pain in the previous 24 h. All patients received aspirin and were randomly assigned treatment with tirofiban or heparin. We took baseline measurements of troponin I and troponin T. We recorded death, myocardial infarction, or recurrent ischaemia after 48 h infusion treatment and at 7 days and 30 days. FINDINGS 629 (28.3%) patients had troponin I concentrations higher than the diagnostic threshold of 1.0 microg/L and 644 (29.0%) troponin T concentrations higher than 0.1 microg/L. 30-day event rates (death, myocardial infarction) were 13.0% for troponin-I-positive patients compared with 4.9% for troponin-I-negative patients (p<0.0001), and 13.7% compared wth 3.5% for troponin T (p<0.001). At 30 days, in troponin-I-positive patients, tirofiban had lowered the risk of death (adjusted hazard ratio 0.25 [95% CI 0.09-0.68], p=0.004) and myocardial infarction (0.37 [0.16-0.84], p=0.01). This benefit was seen in medically managed patients (0.30 [0.10-0.84], p=0.004) and those undergoing revascularisation (0.37 [0.15-0.93] p=0.02) after 48 h infusion treatment. By contrast, no treatment effect was seen for troponin-I-negative patients. Similar benefits were seen for troponin-T-positive patients. INTERPRETATION Troponin I and troponin T reliably identified high-risk patients with acute coronary syndromes, managed medically and by revascularisation, who would benefit from tirofiban.


Ubiquitin functions by covalently modifying other proteins. In the past few years, a surprising number of other proteins have been identified that, despite often being only slightly similar to ubiquitin, can also be attached to proteins. Newly discovered parallels between the activation of ubiquitin and the biosynthesis of certain enzyme cofactors now hint at the possible evolutionary origins of the ubiquitin system.


IMPORTANCE Exacerbations of respiratory symptoms in chronic obstructive pulmonary disease (COPD) have profound and long-lasting adverse effects on patients. OBJECTIVE To test the hypothesis that elevated levels of inflammatory biomarkers in individuals with stable COPD are associated with an increased risk of having exacerbations. DESIGN, SETTING, AND PARTICIPANTS Prospective cohort study examining 61,650 participants with spirometry measurements from the Copenhagen City Heart Study (2001-2003) and the Copenhagen General Population Study (2003-2008). Of these, 6574 had COPD, defined as a ratio between forced expiratory volume in 1 second (FEV1) and forced vital capacity below 0.7. MAIN OUTCOMES AND MEASURES Baseline levels of C-reactive protein (CRP) and fibrinogen and leukocyte count were measured in participants at a time when they were not experiencing symptoms of exacerbations. Exacerbations were recorded and defined as short-course treatment with oral corticosteroids alone or in combination with an antibiotic or as a hospital admission due to COPD. Levels of CRP and fibrinogen and leukocyte count were defined as high or low according to cut points of 3 mg/L, 14 μmol/L, and 9 ×10(9)/L, respectively. RESULTS During follow-up, 3083 exacerbations were recorded (mean, 0.5/participant). In the first year of follow-up, multivariable-adjusted odds ratios for having frequent exacerbations were 1.2 (95% CI, 0.7-2.2; 17 events/1000 person-years) for individuals with 1 high biomarker, 1.7 (95% CI, 0.9-3.2; 32 events/1000 person-years) for individuals with 2 high biomarkers, and 3.7 (95% CI, 1.9-7.4; 81 events/1000 person-years) for individuals with 3 high biomarkers compared with individuals who had no elevated biomarkers (9 events/1000 person-years; trend: P = 2 × 10(-5)). Corresponding hazard ratios using maximum follow-up time were 1.4 (95% CI, 1.1-1.8), 1.6 (95% CI, 1.3-2.2), and 2.5 (95% CI, 1.8-3.4), respectively (trend: P = 1 × 10(-8)). The addition of inflammatory biomarkers to a basic model including age, sex, FEV1 percent predicted, smoking, use of any inhaled medication, body mass index, history of previous exacerbations, and time since most recent prior exacerbation improved the C statistics from 0.71 to 0.73 (comparison: P = 9 × 10(-5)). Relative risks were consistent in those with milder COPD, in those with no history of frequent exacerbations, and in the 2 studies separately. The highest 5-year absolute risks of having frequent exacerbations in those with 3 high biomarkers (vs no high biomarkers) were 62% (vs 24%) for those with Global Initiative for Chronic Obstructive Lung Disease (GOLD) grades C-D (n = 558), 98% (vs 64%) in those with a history of frequent exacerbations (n = 127), and 52% (vs 15%) for those with GOLD grades 3-4 (n = 465). CONCLUSIONS AND RELEVANCE Simultaneously elevated levels of CRP and fibrinogen and leukocyte count in individuals with COPD were associated with increased risk of having exacerbations, even in those with milder COPD and in those without previous exacerbations. Further investigation is needed to determine the clinical value of these biomarkers for risk stratification.


Over the last decade, gene supplementation therapy for inherited retinal degeneration has come of age. Early proof-of-concept studies in animal models of disease showed modest, but genuine improvements in retinal function and/or survival. Further development of the vectors used for gene transfer to the retina has led to better treatment efficacy in a wide variety of animal models, leading in 2008 to the initiation of three clinical trials for Leber congenital amaurosis caused by retinal pigment epithelium 65 deficiency. The results from these trials suggest that the treatment of inherited retinal dystrophy by gene therapy can be safe and effective. Here, we examine the progress of gene supplementation therapy in the retina, and discuss the potential for using gene therapy to treat different forms of inherited retinal degeneration.


Anorectic drugs have been used for more than 30 years as an aid in weight reduction for obese persons. The use of aminorex, an amphetamine analog that increases norepinephrine levels in the central nervous system, led to an epidemic of primary pulmonary hypertension (PPH) in Europe in the late 1960s and early 1970s. The use of fenfluramine and later dexfenfluramine [drugs that inhibit 5-hydroxytryptamine (5-HT) release and reuptake and increases 5-HT and thus 5-HT secretion in the brain] was associated with a second epidemic of PPH. All of these drugs have been voluntarily withdrawn from the market. The pathogenesis of PPH in patients treated with these agents is uncertain, but recent evidence suggests that potassium channel abnormalities and vasoactive and proliferative properties of 5-HT may play a role. There is increasing experimental evidence suggesting that aminorex, fenfluramine and dexfenfluramine inhibit 4-aminopyridine-sensitive currents in potassium channels resulting in vasoconstriction in pulmonary resistance vessels and perhaps smooth muscle cell proliferation. 5-HT causes pulmonary artery vasoconstriction and smooth muscle cell proliferation. Its levels are known to be high in those with fenfluramine-induced PPH. However, a firm cause-and-effect relationship has not yet been established. One potentially beneficial effect of the epidemics of anorectic-related PPH is that it may have provided important insights into the causes of PPH unrelated to anorectic agents.


Dysregulated Wnt signaling is seen in approximately 30% of hepatocellular carcinomas; thus, finding pathways downstream of the activation of Wnt signaling is key. Here, using cre-lox technology, we deleted the Apc gene in the adult mouse liver and observed a rapid increase in nuclear beta-catenin and c-Myc, which is associated with an induction of proliferation that led to hepatomegaly within 4 days of gene deletion. To investigate the downstream pathways responsible for these phenotypes, we analyzed the impact of inactivating APC in the context of deficiency of the potentially key effectors beta-catenin and c-Myc. beta-catenin loss rescues both the proliferation and hepatomegaly phenotypes after APC loss. However, c-Myc deletion, which rescues the phenotypes of APC loss in the intestine, had no effect on the phenotypes of APC loss in the liver. The consequences of the deregulation of the Wnt pathway within the liver are therefore strikingly different from those observed within the intestine, with the vast majority of Wnt targets being beta-catenin-dependent but c-Myc-independent in the liver.


Dry beans and soybeans are nutrient-dense, fiber-rich, and are high-quality sources of protein. Protective and therapeutic effects of both dry bean and soybean intake have been documented. Studies show that dry bean intake has the potential to decrease serum cholesterol concentrations, improve many aspects of the diabetic state, and provide metabolic benefits that aid in weight control. Soybeans are a unique source of the isoflavones genistein and diadzein, which have numerous biological functions. Soybeans and soyfoods potentially have multifaceted health-promoting effects, including cholesterol reduction, improved vascular health, preserved bone mineral density, and reduction of menopausal symptoms. Soy appears to have salutary effects on renal function, although these effects are not well understood. Whereas populations consuming high intakes of soy have lower prevalences of certain cancers, definitive experimental data are insufficient to clarify a protective role of soy. The availability of legume products and resources is increasing, incorporating dry beans and soyfoods into the diet can be practical and enjoyable. With the shift toward a more plant-based diet, dry beans and soy will be potent tools in the treatment and prevention of chronic disease.


A change in the probability of neurotransmitter release (Pr) is an important mechanism underlying synaptic plasticity. Although Pr is often assumed to be the same for all terminals at a single synapse, this assumption is difficult to reconcile with the nonuniform size and structure of synaptic terminals in the central nervous system. Release probability was measured at excitatory synapses on cultured hippocampal neurons by analysis of the progressive block of N-methyl-D-aspartate receptor-mediated synaptic currents by the irreversible open channel blocker MK-801. Release probability was nonuniform (range of 0.09 to 0.54) for terminals arising from a single axon, the majority of which had a low Pr. However, terminals with high Pr are more likely to be affected by the activity-dependent modulation that occurs in long-term potentiation.


Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is the only described systemic autoimmune disease with established monogenic background, and the first autoimmune disorder localized outside the major histocompatibility complex (MHC) region. The primary biochemical defect in APECED is unknown. We have isolated a novel gene, AIRE, encoding for a putative nuclear protein featuring two PHD-type zinc-finger motifs, suggesting its involvement in transcriptional regulation. Five mutations in AIRE are reported in individuals with this disorder. This is the first report of a single-gene defect causing a systemic human autoimmune disease, providing a tool for exploring the molecular basis of autoimmunity.


Targeting mitotic exit has been recently proposed as a relevant therapeutic approach against cancer. By using genetically engineered mice, we show that the APC/C cofactor Cdc20 is essential for anaphase onset in vivo in embryonic or adult cells, including progenitor/stem cells. Ablation of Cdc20 results in efficient regression of aggressive tumors, whereas current mitotic drugs display limited effects. Yet, Cdc20 null cells can exit from mitosis upon inactivation of Cdk1 and the kinase Mastl (Greatwall). This mitotic exit depends on the activity of PP2A phosphatase complexes containing B55α or B55δ regulatory subunits. These data illustrate the relevance of critical players of mitotic exit in mammals and their implications in the balance between cell death and mitotic exit in tumor cells.


DrugBank (www.drugbank.ca) is a web-enabled database containing comprehensive molecular information about drugs, their mechanisms, their interactions and their targets. First described in 2006, DrugBank has continued to evolve over the past 12 years in response to marked improvements to web standards and changing needs for drug research and development. This year's update, DrugBank 5.0, represents the most significant upgrade to the database in more than 10 years. In many cases, existing data content has grown by 100% or more over the last update. For instance, the total number of investigational drugs in the database has grown by almost 300%, the number of drug-drug interactions has grown by nearly 600% and the number of SNP-associated drug effects has grown more than 3000%. Significant improvements have been made to the quantity, quality and consistency of drug indications, drug binding data as well as drug-drug and drug-food interactions. A great deal of brand new data have also been added to DrugBank 5.0. This includes information on the influence of hundreds of drugs on metabolite levels (pharmacometabolomics), gene expression levels (pharmacotranscriptomics) and protein expression levels (pharmacoprotoemics). New data have also been added on the status of hundreds of new drug clinical trials and existing drug repurposing trials. Many other important improvements in the content, interface and performance of the DrugBank website have been made and these should greatly enhance its ease of use, utility and potential applications in many areas of pharmacological research, pharmaceutical science and drug education.


The development of in vitro assays to analyze pre-mRNA splicing resulted in the discovery of many fundamental features characterizing splicing signals and the machinery that completes this process. Because in vitro assays can be manipulated by various biochemical approaches, the versatility of investigating alternative pre-mRNA splicing in the test tube appears endless. Importantly, modifications in reaction conditions can lead to the accumulation, isolation, and characterization of reaction intermediates, a prerequisite for gaining mechanistic insights into how the spliceosome carries out intron removal, and how regulatory elements assist the general splicing machinery in defining splice sites and alternative exons. These considerable experimental advantages have made the in vitro splicing system a standard assay, even though this approach is independent from RNA transcription and other RNA processing events, and in some respects deviates from the natural process of mRNA biogenesis. Here, we describe the tools and techniques necessary to carry out in vitro splicing assays. Analyses of various experimental designs are presented to highlight the approaches taken to gain insights into the mechanisms by which splice site recognition and activation are communicated with the general splicing machinery. Methods to measure the kinetics of splicing, to observe the formation of the pre-spliceosomal complexes, and to manipulate and modify the in vitro system to resolve the regulatory influences in alternative splicing are presented.


We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.


BACKGROUND Obesity is an epidemic that affects 1 in 3 individuals in the United States, and recent evidence suggests that enteric microbiota may play a significant role in the development of obesity. This study evaluated the association between methanogenic archaea and obesity in human subjects. METHODS Subjects with a body mass index (BMI) of 30 kg/m² or higher were prospectively recruited from the weight loss program of a tertiary care medical center. Subjects who met the study's inclusion criteria were asked to complete a questionnaire that included a series of visual analogue scores for bowel symptom severities. Subjects then provided a single end-expiratory breath sample to quantitate methane levels. Bivariate and multivariate analyses were used to determine associations with BMI. RESULTS A total of 58 patients qualified for enrollment. Twenty percent of patients (n = 12) had breath test results that were positive for methane (>3 parts per million [ppm]), with a mean breath methane concentration of 12.2±3.1 ppm. BMI was significantly higher in methane-positive subjects (45.2±2.3 kg/m²) than in methane-negative subjects (38.5±0.8 kg/m²; P=.001). Methane-positive subjects also had a greater severity of constipation than methane-negative subjects (21.3±6.4 vs 9.5±2.4; P=.043). Multiple regression analysis illustrated a significant association between BMI and methane, constipation, and antidepressant use. However, methane remained an independent predictor of elevated BMI when controlling for antidepressant use (P<.001) and when controlling for both constipation and antidepressant use (6.55 kg/m² greater BMI; P=.003). CONCLUSION This is the first human study to demonstrate that a higher concentration of methane detected by breath testing is a predictor of significantly greater obesity in overweight subjects.


BACKGROUND Venous thromboembolism (VTE) is a common complication in cancer patients and a significant cause of morbidity and mortality. However, little information is available on oncologists' perceptions of the risk of VTE and its management. The Fundamental Research in Oncology and Thrombosis (FRONTLINE) study is the first comprehensive global survey of thrombosis and cancer. The study was designed to collect data on the perceived risk and patterns of practice with regard to VTE in cancer patients undergoing surgical and medical management of their malignancy and to provide information on international and regional practice patterns, allowing for the design of research studies to answer the concerns of practicing clinicians. METHODS Literature reviews were performed to provide a current evidence base against which to compare the findings, and a survey was developed under the guidance of an advisory board. A paper-based reply-paid questionnaire was distributed globally between July and November 2001 to clinicians involved in cancer care and was made available on a dedicated website. FINDINGS A total of 3,891 completed responses were available for analysis. Brain and pancreatic tumors were considered to carry a high risk for VTE, and 80% of respondents considered the use of central venous lines to be associated with a high risk of VTE. Marked differences were seen in the use of thromboprophylaxis for surgical and medical cancer patients, with over 50% of surgeons reporting that they initiated thromboprophylaxis routinely, while most medical oncologists reported using thromboprophylaxis in less than 5% of medical patients. Low molecular weight heparin (LMWH) was the most popular method of thromboprophylaxis employed in both surgical and medical patients and was more favored by European than U.S. clinicians. Some 20% of respondents reported using aspirin for prophylaxis, despite there being no reliable evidence for this agent as effective in prevention in this population. For the treatment of VTE, LMWH was again the most common initial treatment, although, for the long-term, oral anticoagulation therapy was widely adopted. Many patients were treated for VTE on an outpatient basis, and secondary prevention of VTE was typically continued for 3 to 6 months after an episode of deep vein thrombosis or for longer in the case of pulmonary embolism. INTERPRETATION The results of the FRONTLINE survey demonstrate a need for guidelines to direct clinical practice in line with evidence-based data concerning cancer and VTE. Oncologists need to be educated regarding the true risks of VTE associated with certain cancers and on strategies for prevention and treatment to reduce the morbidity and mortality associated with VTE in all cancer patients. The study has also helped identify areas for future research.


BACKGROUND Histone deimination regulates gene function and contributes to antimicrobial response, allowing the formation of neutrophil extracellular traps (NETs). Deiminated proteins are target of anti-citrullinated peptides antibodies (ACPA) in rheumatoid arthritis (RA). OBJECTIVE The objective of this paper is to test the hypothesis that RA sera react with deiminated histones contained in NETs. METHODS Neutrophils from peripheral blood were stimulated with A23187 and acid treated; NETosis was induced by phorbol myristate acetate, and NET proteins were isolated. Sera were tested by immunoblot on acid extracted proteins from neutrophils and from NETs, and by ELISA on deiminated histone H4 or H4-derived peptides. Bands reactive with RA sera were excised from gels, digested with trypsin and subjected to matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) analysis, before and after derivatisation to detect citrullinated peptides. RESULTS RA sera reacted with a deiminated antigen of 11 KDa from activated neutrophils, recognised also by anti-H4 and antideiminated H4 antibodies. A similar reactivity was observed with NET proteins. The antigen from neutrophils or NETs was identified as citrullinated H4 by MALDI-TOF analysis. By ELISA, RA sera bound in vitro citrullinated H4. Citrullinated H4 14-34 and 31-50 peptides detected antibodies in 67% and 63% of RA sera and in less than 5% of controls; antibody titre was correlated with anti-CCP2. CONCLUSIONS Citrullinated H4 from activated neutrophils and NETs is a target of antibodies in RA, and synthetic citrullinated H4-derived peptides are a new substrate for ACPA detection. As NETosis can generate antigens for ACPA, these data suggest a novel connection between innate and adaptive immunity in RA.


CONTEXT Managing thoracic aortic aneurysms identified incidentally by increased use of computed tomography, echocardiography, and magnetic resonance imaging is problematic, especially in the elderly. OBJECTIVE To ascertain whether the previously reported poor prognosis for individuals with thoracic aortic aneurysms has changed with better medical therapies and improved surgical techniques that can now be applied to aneurysm management. DESIGN Population-based cohort study. SETTING AND PATIENTS All 133 patients with the diagnosis of degenerative thoracic aortic aneurysms among Olmsted County, Minnesota, residents between 1980 and 1994 compared with a previously reported cohort of similar patients between 1951 and 1980. MAIN OUTCOME MEASURES The primary clinical end points were incidence, cumulative rupture risk, rupture risk as a function of aneurysm size, and survival. RESULTS In contrast to abdominal aortic aneurysms, for which men are affected predominately, 51% of thoracic aortic aneurysms were identified in women who were considerably older at recognition than men (mean age, 75.9 vs 62.8 years, respectively; P= .01). The overall incidence rate of 10.4 per 100000 person-years (95% confidence interval [CI], 8.6-12.2) between 1980 and 1994 was more than 3-fold higher than the rate from 1951 to 1980. The cumulative risk of rupture was 20% after 5 years. Seventy-nine percent of ruptures occurred in women (P= .01). The 5-year risk of rupture as a function of aneurysm size at recognition was 0% for aneurysms less than 4 cm in diameter, 16% (95% CI, 4%-28%) for those 4 to 5.9 cm, and 31% (95% CI, 5%-56%) for aneurysms 6 cm or more. Overall 5-year survival improved to 56% (95% CI, 48%-66%) between 1980 and 1994 compared with only 19% between 1951 and 1980 (P<.01). CONCLUSIONS In this population, elderly women represent an increasing portion of all patients with clinically recognized thoracic aortic aneurysms and constitute the majority of patients whose aneurysm eventually ruptures. Overall survival for thoracic aortic aneurysms has improved significantly in the past 15 years.


DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.


Progressive advances using zebrafish as a model organism have provided hematologists with an additional genetic system to study blood cell formation and hematological malignancies. Despite extensive evolutionary divergence between bony fish (teleosts) and mammals, the molecular pathways governing hematopoiesis have been highly conserved. As a result, most (if not all) of the critical hematopoietic transcription factor genes identified in mammals have orthologues in zebrafish. As in other vertebrates, all of the teleost blood lineages are believed to originate from a pool of pluripotent, self-renewing hematopoietic stem cells. Here, we provide a detailed review of the timing, anatomical location, and transcriptional regulation of zebrafish ‘primitive’ and ‘definitive’ hematopoiesis as well as discuss a model of T-cell leukemia and recent advances in blood cell transplantation. Given that many of the regulatory genes that control embryonic hematopoiesis have been implicated in oncogenic pathways in adults, an understanding of blood cell ontogeny is likely to provide insights into the pathophysiology of human leukemias.


We have provided a historical perspective on a body of steroid receptor research dealing with the structure and physiological significance of the untransformed 9S receptor that has often confused both novice and expert investigators. The frequent controversies and equivocations of earlier studies were due to the fact that the native, hormone-free state of these receptors is a large multiprotein complex that resisted description for many years because of its unstable and dynamic nature. The untransformed 9S state of the steroid and dioxin receptors has provided a unique system for studying the function of the ubiquitous, abundant, and conserved heat shock protein, hsp90. The hormonal control of receptor association with hsp90 provided a method of manipulating the receptor heterocomplex in a manner that was physiologically meaningful. For several steroid receptors, binding to hsp90 was required for the receptor to be in a native hormone-binding state, and for all of the receptors, hormone binding promoted dissociation of the receptor from hsp90 and conversion of the receptor to the DNA-binding state. Although the complexes between tyrosine kinases and hsp90 were discovered earlier, the hormonal regulation or steroid receptor association with hsp90 permitted much more rapid and facile study of hsp90 function. The observations that hsp90 binds to the receptors through their HBDs and that these domains can be fused to structurally different proteins bringing their function under hormonal control provided a powerful linkage between the hormonal regulation of receptor binding to hsp90 and the initial step in steroid hormone action. Because the 9S receptor hsp90 heterocomplexes could be physically stabilized by molybdate, their protein composition could be readily studied, and it became clear that these complexes are multiprotein structures containing a number of unique proteins, such as FKBP51, FKBP52, CyP-40, and p23, that were discovered because of their presence in these structures. Further analysis showed that hsp90 itself exists in a variety of native multiprotein heterocomplexes independent of steroid receptors and other 'substrate' proteins. Cell-free systems can now be used to study the formation of receptor heterocomplexes. As we outlined in the scheme of Fig. 1, the multicomponent receptor-hsp90 heterocomplex assembly system is being reconstituted, and the importance of individual proteins, such as hsp70, p60, and p23, in the assembly process is becoming recognized. It should be noted that our understanding of the mechanism and purpose of steroid receptor heterocomplex assembly is still at an early stage. We can now speculate on the roles of receptor-associated proteins in receptor action, both as individuals and as a group, but their actual functions are still vague or unknown. We can make realistic models about the chaperoning and trafficking of steroid receptors, but we don't yet know how these processes occur, we don't know where chaperoning occurs in the cell (e.g. Is it limited to the cytoplasm? Is it a diffuse process or does chaperoning occur in association with structural elements?), and, with the exception of the requirement for hormone binding, we don't know the extent to which the hsp90-based chaperone system impacts on steroid hormone action. It is not yet clear how far the discovery of this hsp90 heterocomplex assembly system will be extended to the development of a general understanding of protein processing in the cell. Because this assembly system is apparently present in all eukaryotic cells, it probably performs an essential function for many proteins. The bacterial homolog of hsp90 is not an essential protein, but hsp90 is essential in eukaryotes, and recent studies indicate that the development of the cell nucleus from prokaryotic progenitors was accompanied by the duplication of genes for hsp90 and hsp70 (698). (ABSTRACT TRUNCATED)


Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.


The EUROCARE project analysed cancer survival data from 45 population-based cancer registries in 17 European countries, revealing wide international differences in cancer survival. We calculated 5-year relative survival for 1836287 patients diagnosed with one of 13 cancers during the period 1978-1989. The data, from 20 cancer registries in 13 countries, were grouped into four regions: Finland, Sweden, Iceland (Northern Europe); Denmark, England and Scotland (UK and Denmark); France, The Netherlands, Germany, Italy and Switzerland (Western Europe); Estonia and Poland (Eastern Europe), and broken down into four periods (1978-1980, 1981-1983, 1984-1986, 1987-1989). For each cancer, mean European and regional survival was estimated as the weighted mean of 5-year relative survival in each country. Survival increased with time for all tumours, particularly for cancers of testis (12% increase, i.e. from 79.9 to 91.9%), breast, large bowel, skin melanoma (approximately 9-10%), and lymphomas (approximately 7%). For most solid tumours, survival was highest in Northern Europe and lowest in Eastern Europe, and also low in the UK and Denmark. Regional variation was less marked for the lymphomas. Survival improved more in Western than Northern Europe, and the differences between these regions fell for bowel cancer (from 8.0% for those diagnosed in 1978-1980 to 2% for those diagnosed in 1987-1989), breast cancer (from 7.4% to 3.9%), skin melanoma (from 13.4% to 11.0%) and Hodgkin's disease (from 7.2 to 0.6%). For potentially curable malignancies such as Hodgkin's disease, large bowel, breast and testicular cancers, there were substantial increases in survival, suggesting an earlier diagnosis and more effective treatment. The persisting regional differences suggest there are corresponding differences in the availability of diagnostic and therapeutic facilities, and in the effectiveness of healthcare systems.


In eukaryotes, a surveillance mechanism known as nonsense-mediated decay (NMD) degrades the mRNA when a premature-termination codon (PTC) is present. NMD requires translation to read the frame of the mRNA and detect the PTC. During pre-mRNA splicing, the exon-exon junction complex (EJC) is recruited to a region 20-24 nt upstream of the exon junction on the mature mRNA. The presence of a PTC upstream from the EJC elicits NMD. Eukaryotic initiation factor 4A (eIF4A) III is a nuclear protein that interacts physically or functionally with translation initiation factors eIF4G and eIF4B, respectively, and shares strikingly high identity with the initiation factors eIF4AI/II. Here we show that siRNA against eIF4AIII, but not against eIF4AI/II, inhibits NMD. Moreover, eIF4AIII, but not eIF4AI, is specifically recruited to the EJC during splicing. The observations that eIF4AIII is loaded onto the mRNA during splicing in the nucleus, has properties related to a translation initiation factor, and functions in NMD raises the possibility that eIF4AIII substitutes for eIF4AI/II during NMD.


Swaddling was an almost universal child-care practice before the 18th century. It is still tradition in certain parts of the Middle East and is gaining popularity in the United Kingdom, the United States, and The Netherlands to curb excessive crying. We have systematically reviewed all articles on swaddling to evaluate its possible benefits and disadvantages. In general, swaddled infants arouse less and sleep longer. Preterm infants have shown improved neuromuscular development, less physiologic distress, better motor organization, and more self-regulatory ability when they are swaddled. When compared with massage, excessively crying infants cried less when swaddled, and swaddling can soothe pain in infants. It is supportive in cases of neonatal abstinence syndrome and infants with neonatal cerebral lesions. It can be helpful in regulating temperature but can also cause hyperthermia when misapplied. Another possible adverse effect is an increased risk of the development of hip dysplasia, which is related to swaddling with the legs in extension and adduction. Although swaddling promotes the favorable supine position, the combination of swaddling with prone position increases the risk of sudden infant death syndrome, which makes it necessary to warn parents to stop swaddling if infants attempt to turn. There is some evidence that there is a higher risk of respiratory infections related to the tightness of swaddling. Furthermore, swaddling does not influence rickets onset or bone properties. Swaddling immediately after birth can cause delayed postnatal weight gain under certain conditions, but does not seem to influence breastfeeding parameters.


The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here, we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (Ndp(AP)). In the CNS, Ndp(AP) expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of Ndp(AP) expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, Ndp(AP) expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea.


Excitation-transcription coupling, linking stimulation at the cell surface to changes in nuclear gene expression, is conserved throughout eukaryotes. How closely related coexpressed transcription factors are differentially activated remains unclear. Here, we show that two Ca2+-dependent transcription factor isoforms, NFAT1 and NFAT4, require distinct sub-cellular InsP3 and Ca2+ signals for physiologically sustained activation. NFAT1 is stimulated by sub-plasmalemmal Ca2+ microdomains, whereas NFAT4 additionally requires Ca2+ mobilization from the inner nuclear envelope by nuclear InsP3 receptors. NFAT1 is rephosphorylated (deactivated) more slowly than NFAT4 in both cytoplasm and nucleus, enabling a more prolonged activation phase. Oscillations in cytoplasmic Ca2+, long considered the physiological form of Ca2+ signaling, play no role in activating either NFAT protein. Instead, effective sustained physiological activation of NFAT4 is tightly linked to oscillations in nuclear Ca2+. Our results show how gene expression can be controlled by coincident yet geographically distinct Ca2+ signals, generated by a freely diffusible InsP3 message.


Cerebral apolipoprotein E (apoE) has been implicated in neuronal protection and repair. Due to the variable levels and types of estrogen receptors within different brain regions, the effect of estrogen on apoE and the mechanism of this effect may vary within different regions. Ovariectomized female C57BL/6 mice were treated with pharmacological levels of 17 beta-estradiol or placebo for 5 days, resulting in supraphysiological plasma levels of estradiol in the treated mice. ApoE and glial fibrillary acidic protein (GFAP) levels were measured in the cortex, hippocampus and diencephalon. 17 beta-Estradiol up-regulated apoE but not GFAP in the cortex and diencephalon, whereas in the hippocampus, GFAP and apoE were equally up-regulated. Treatment of estrogen receptor (ER) alpha knockout mice with 17 beta-estradiol or treatment of C57BL/6 mice with 17 alpha-estradiol, a poor estrogen receptor agonist, specifically induced apoE in the cortex, but not in the diencephalon. These results indicate that 17 beta-estradiol effects on apoE are either directly or indirectly mediated by ER alpha in the diencephalon, while the effects in the cortex may be mediated by a non-classical mechanism or by ER beta. Measurement of mRNA levels in estrogen versus placebo-treated wild-type mice indicated that the effect of 17 beta-estradiol on apoE was not associated with changes in apoE mRNA levels.


Hepatitis C virus (HCV) is a single-stranded RNA virus encoding a single polyprotein whose translation is driven by an internal ribosome entry site (IRES). HCV infection strongly induces antiviral interferon-stimulated gene (ISG) expression in the liver, yet it persists, suggesting that HCV can block ISG effector function. We now show that HCV infection triggers phosphorylation and activation of the RNA-dependent protein kinase PKR, which inhibits eukaryotic translation initiation factor eIF2 alpha and attenuates ISG protein expression despite normal ISG mRNA induction. ISG protein induction is restored and the antiviral effects of interferon are enhanced when PKR expression is suppressed in interferon-treated infected cells. Whereas host protein translation, including antiviral ISGs, is suppressed by activated PKR, HCV IRES-dependent translation is not. These results suggest that the ability of HCV to activate PKR may, paradoxically, be advantageous for the virus during an IFN response by preferentially suppressing the translation of ISGs.


The aim of the present research is to ascertain whether in larval Xenopus laevis nerve-independence for the regeneration of early stage limbs and nerve-dependence of late stage limbs observed in a previous work (Filoni and Paglialunga, '90) is related to extrinsic (systemic) factors or to intrinsic changes taking place in the limb cells themselves during development. In this paper the regenerative capacity of early and late stage hindlimbs under the same extrinsic conditions, insofar as both are grafted onto the denervated hindlimbs of host larvae at the same developmental stage, is studied. All the grafted limbs are amputated after the host larvae have reached stage 57-58 (according to Nieuwkoop and Faber, '56). In experiment I, the grafted limb is amputated at stage 52, at the thigh level; in experiment II, the grafted limb is amputated at stage 54-55, at the tarsalia level; in experiment III the grafted limb is amputated at stage 57, at the tarsalia level. In all three experiments, together with the grafted limb, also the host limb is amputated at the tarsalia level. The results show that while grafted limbs amputated at stages 52 and 54-55 regenerate in the absence of nerves, grafted limbs amputated at stage 57 cannot. The failure of late stage grafted limbs to regenerate cannot be explained in terms of an immune-type inhibiting reaction since it has been observed also in denervated autografted limbs and in the host limbs. Since all the grafted limbs are in the same environmental conditions, the results show that in larval Xenopus laevis nerve-independence for regeneration of early stage limbs and nerve-dependence of late stage limbs are not related to factors extrinsic to the limb but to intrinsic changes taking place in the limb cells themselves during development.


The olfactory epithelium houses chemosensory neurons, which transmit odor information from the nose to the brain. In adult mammals, the olfactory epithelium is a uniquely robust neuroproliferative zone, with the ability to replenish its neuronal and non-neuronal populations due to the presence of germinal basal cells. The stem and progenitor cells of these germinal layers, and their regulatory mechanisms, remain incompletely defined. Here we show that progenitor cells expressing c-Kit, a receptor tyrosine kinase marking stem cells in a variety of embryonic tissues, are required for maintenance of the adult neuroepithelium. Mouse genetic fate-mapping analyses show that embryonically, a c-Kit(+) population contributes to olfactory neurogenesis. In adults under conditions of normal turnover, there is relatively sparse c-Kit(+) progenitor cell (ckPC) activity. However, after experimentally induced neuroepithelial injury, ckPCs are activated such that they reconstitute the neuronal population. There are also occasional non-neuronal cells found to arise from ckPCs. Moreover, the selective depletion of the ckPC population, utilizing temporally controlled targeted diphtheria toxin A expression, results in failure of neurogenesis after experimental injury. Analysis of this model indicates that most ckPCs reside among the globose basal cell populations and act downstream of horizontal basal cells, which can serve as stem cells. Identification of the requirement for olfactory c-Kit-expressing progenitors in olfactory maintenance provides new insight into the mechanisms involved in adult olfactory neurogenesis. Additionally, we define an important and previously unrecognized site of adult c-Kit activity.


Amyotrophic Lateral Sclerosis (ALS) is a progressive degenerative  disease of the motor neurons and the cause is unknown. Diverse factors such as  genetic defects, nutritional deficiencies, head trauma, environmental toxin,  autoimmune responses and viral and bacterial infections are involved. Mycoplasmas  have been implicated as causal agents of different illnesses in human. The  purpose of this study was to investigate the presence of mycoplasmas in  the bloodstream of patients with ALS. Patients with ALS and healthy individuals  were included in the study. A blood sample was taken in tubes with or without  anticoagulant. Mycoplasmas were detected by culture or direct PCR, and the  presence of antibodies IgM and IgG against LAMPs of these microorganisms by  Western blot. Cultures for aerobic facultative bacteria were also done. Blood samples  from 13 patients and 44 healthy individuals were screened. All blood cultures  for non-fermentative mycoplasmas and aerobic facultative bacteria were  negative. Cultures for fermentative mycoplasmas were considered positive after  identification of mycoplasmal DNA by PCR. Mycoplasma sp. was detected by culture or  direct PCR in 6/13 (46%) patients and in 4/44 (9%) of healthy individuals. M. fermentans was detected by PCR using  specific primers in six patients and in two healthy individuals. IgM against  LAMPs of M. fermentans were detected  in 6/13 (46%) blood samples from patients and in 13/44 (30%) from healthy  individuals, while. IgG was detected in 4/13 (31%) patients and in 3/44 (7%)  healthy individuals. The results of this study show that mycoplasmas cause a  systemic infection and could play a role in the origin or progression of the  ALS.


Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.


The role of transient receptor potential M4 (Trpm4), an unusual member of the Trp family of ion channels, is poorly understood. Using rodent models of spinal cord injury, we studied involvement of Trpm4 in the progressive expansion of secondary hemorrhage associated with capillary fragmentation, the most destructive mechanism of secondary injury in the central nervous system. Trpm4 mRNA and protein were abundantly upregulated in capillaries preceding their fragmentation and formation of petechial hemorrhages. Trpm4 expression in vitro rendered COS-7 cells highly susceptible to oncotic swelling and oncotic death following ATP depletion. After spinal cord injury, in vivo gene suppression in rats treated with Trpm4 antisense or in Trpm4−/− mice preserved capillary structural integrity, eliminated secondary hemorrhage, yielded a threefold to fivefold reduction in lesion volume and produced a substantial improvement in neurological function. To our knowledge, this is the first example of a Trp channel that must undergo de novo expression for manifestation of central nervous system pathology.


CONTEXT Vascular stiffness increases with advancing age and is a major risk factor for age-related morbidity and mortality. Vascular stiffness and blood pressure pulsatility are related; however, temporal relationships between vascular stiffening and blood pressure elevation have not been fully delineated. OBJECTIVE To examine temporal relationships among vascular stiffness, central hemodynamics, microvascular function, and blood pressure progression. DESIGN, SETTING, AND PARTICIPANTS Longitudinal community-based cohort study conducted in Framingham, Massachusetts. The present investigation is based on the 2 latest examination cycles (cycle 7: 1998-2001; cycle 8: 2005-2008 [last visit: January 25, 2008]) of the Framingham Offspring study (recruited: 1971-1975). Temporal relationships among blood pressure and 3 measures of vascular stiffness and pressure pulsatility derived from arterial tonometry (carotid-femoral pulse wave velocity [CFPWV], forward wave amplitude [FWA], and augmentation index) were examined over a 7-year period in 1759 participants (mean [SD] age: 60 [9] years; 974 women). MAIN OUTCOME MEASURES The primary outcomes were blood pressure and incident hypertension during examination cycle 8. The secondary outcomes were CFPWV, FWA, and augmentation index during examination cycle 8. RESULTS In a multivariable-adjusted regression model, higher FWA (β, 1.3 [95% CI, 0.5-2.1] mm Hg per 1 SD; P = .002) and higher CFPWV (β, 1.5 [95% CI, 0.5-2.6] mm Hg per 1 SD; P = .006) during examination cycle 7 were jointly associated with systolic blood pressure during examination cycle 8. Similarly, in a model that included systolic and diastolic blood pressure and additional risk factors during examination cycle 7, higher FWA (odds ratio [OR], 1.6 [95% CI, 1.3-2.0] per 1 SD; P < .001), augmentation index (OR, 1.7 [95% CI, 1.4-2.0] per 1 SD; P < .001), and CFPWV (OR, 1.3 [95% CI, 1.0-1.6] per 1 SD; P = .04) were associated with incident hypertension during examination cycle 8 (338 cases [32%] in 1048 participants without hypertension during examination cycle 7). Conversely, blood pressure during examination cycle 7 was not associated with CFPWV during examination cycle 8. Higher resting brachial artery flow (OR, 1.23 [95% CI, 1.04-1.46]) and lower flow-mediated dilation (OR, 0.80 [95% CI, 0.67-0.96]) during examination cycle 7 were associated with incident hypertension (in models that included blood pressure and tonometry measures collected during examination cycle 7). CONCLUSION In this cohort, higher aortic stiffness, FWA, and augmentation index were associated with higher risk of incident hypertension; however, initial blood pressure was not independently associated with risk of progressive aortic stiffening.


The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl. html ) constitutes Europe's primary nucleotide sequence resource. DNA and RNA sequences are directly submitted from researchers and genome sequencing groups and collected from the scientific literature and patent applications (Fig. 1). In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute. Database releases are produced quarterly and are distributed on CD-ROM. EBI's network services allow access to the most up-to-date data collection via Internet and World Wide Web interface, providing database searching and sequence similarity facilities plus access to a large number of additional databases.


Neoplastic cells are genetically unstable. Strategies that target pathways affecting genome instability can be exploited to disrupt tumor cell growth, potentially with limited consequences to normal cells. Chromosomal instability (CIN) is one type of genome instability characterized by mitotic defects that increase the rate of chromosome mis-segregation. CIN is frequently caused by extra centrosomes that transiently disrupt normal bipolar spindle geometry needed for accurate chromosome segregation. Tumor cells survive with extra centrosomes because of biochemical pathways that cluster centrosomes and promote chromosome segregation on bipolar spindles. Recent work shows that targeted inhibition of these pathways prevents centrosome clustering and forces chromosomes to segregate to multiple daughter cells, an event triggering apoptosis that we refer to as anaphase catastrophe. Anaphase catastrophe specifically kills tumor cells with more than 2 centrosomes. This death program can occur after genetic or pharmacologic inhibition of cyclin dependent kinase 2 (Cdk2) and is augmented by combined treatment with a microtubule inhibitor. This proapoptotic effect occurs despite the presence of ras mutations in cancer cells. Anaphase catastrophe is a previously unrecognized mechanism that can be pharmacologically induced for apoptotic death of cancer cells and is, therefore, appealing to engage for cancer therapy and prevention.


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.


Chronic tension-type headache (CTTH) assessed by proband report was evaluated in a family study of CTTH. A clinical interview of first-degree relatives by a physician was used as index of validity. Familial occurrence of CTTH in first-degree relatives was also investigated. Patterns of familial aggregation of CTTH were assessed by calculating the population relative risk. A neurological resident carried out all the interviews of probands and their first-degree relatives. The operational diagnostic criteria of the International Headache Society were used. The 122 probands had 377 first-degree relatives. Sensitivity, specificity, predictive values, and chance-corrected agreement rate for the diagnosis CTTH were 68%, 86%, 53% (PVpos), 92% (PVneg), and 0.48, respectively. The low sensitivity of CTTH assessed by proband report indicates that a clinical interview by a physician is necessary in family studies of CTTH. Clinically interviewed parents, siblings, and children had a 2.1 to 3.9-fold significantly increased risk of CTTH compared with the general population. The gender of the probands did not influence the risk of CTTH among first-degree relatives. The significantly increased familial risk of CTTH and no increased risk of CTTH in spouses suggest that a genetic factor is involved in CTTH.


We have recently isolated SMAP (Smg GDS-associated protein; Smg GDS: small G protein GDP dissociation stimulator) as a novel Smg GDS-associated protein, which has Armadillo repeats and is phosphorylated by Src tyrosine kinase. SMAP is a human counterpart of mouse KAP3 (kinesin superfamily-associated protein) that is associated with mouse KIF3A/B (a kinesin superfamily protein), which functions as a microtubule-based ATPase motor for organelle transport. We isolated here a SMAP-interacting protein from a human brain cDNA library, identified it to be a human homolog of Xenopus XCAP-E (Xenopus chromosome-associated polypeptide), a subunit of condensins that regulate the assembly and structural maintenance of mitotic chromosomes, and named it HCAP (Human chromosome-associated polypeptide). Tissue and subcellular distribution analyses indicated that HCAP was ubiquitously expressed and highly concentrated in the nuclear fraction, where SMAP and KIF3B were also present. SMAP was extracted as a ternary complex with HCAP and KIF3B from the nuclear fraction in the presence of Mg-ATP. The results suggest that SMAP/KAP3 serves as a linker between HCAP and KIF3A/B in the nucleus, and that SMAP/KAP3 plays a role in the interaction of chromosomes with an ATPase motor protein.


Deciphering the molecular basis of stem cell pluripotency is fundamental to the understanding of stem cell biology, early embryonic development, and to the clinical application of regenerative medicine. We report here that the molecular chaperone heat shock protein 90 (Hsp90) is essential for mouse embryonic stem cell (ESC) pluripotency through regulating multiple pluripotency factors, including Oct4, Nanog, and signal transducer and activator of transcription 3. Inhibition of Hsp90 by either 17-N-Allylamino-17-demethoxygeldanamycin or miRNA led to ESC differentiation. Overexpression of Hsp90β partially rescued the phenotype; in particular, the levels of Oct4 and Nanog were restored. Notably, Hsp90 associated with Oct4 and Nanog in the same cellular complex and protected them from degradation by the ubiquitin proteasome pathway, suggesting that Oct4 and Nanog are potential novel Hsp90 client proteins. In addition, Hsp90 inhibition reduced the mRNA level of Oct4, but not that of Nanog, indicating that Hsp90 participates in Oct4 mRNA processing or maturation. Hsp90 inhibition also increased expression of some protein markers for mesodermal lineages, implying that Hsp90 suppresses mesodermal differentiation from ESCs. These findings support a new role for Hsp90 in maintaining ESC pluripotency by sustaining the level of multiple pluripotency factors, particularly Oct4 and Nanog.


Evidence suggests that dormant, microscopic tumors are not only common, but are highly prevalent in otherwise healthy individuals. Due to their small size and non-invasive nature, these dormant tumors remain asymptomatic and, in most cases, undetected. With advances in diagnostic imaging and molecular biology, it is now becoming clear that such neoplasms can remain in an asymptomatic, dormant stage for considerable periods of time without expanding in size. Although a number of processes may play a role in thwarting the expansion of microscopic tumors, one critical mechanism behind tumor dormancy is the ability of the tumor population to induce angiogenesis. Although cancer can arise through multiple pathways, it is assumed that essentially most tumors begin as microscopic, non-angiogenic neoplasms which cannot expand in size until vasculature is established. It is now becoming clear that cancer does not progress through a continuous exponential growth and mass expansion. Clinical cancer is usually manifested only in late, unavoidably symptomatic stages of the disease when tumors are sufficiently large to be readily detected. While dormancy in primary tumors is best defined as the time between the carcinogenic transformation event and the onset of inexorable progressive growth, it can also occur as minimal residual or occult disease from treated tumors or as micro-metastases. The existence of dormant tumors has important implications for the early detection and treatment of cancer. Elucidating the regulatory machinery of these processes will be instrumental in identifying novel early cancer biomarkers and could provide a rationale for the development of dormancy-promoting tumor therapies. Despite the high prevalence of microscopic, dormant tumors in humans and the significant clinical implications of their early detection, this area in cancer research has, to date, been under-investigated. In this mini review observations, models and experimental approaches to study tumor dormancy are summarized. Additionally, analogies and distinctions between the concepts of "tumor dormancy" and that of the "cellular dormancy" of tumor cells, as well as between the "exit from tumor dormancy" and the "onset of the angiogenic switch" are discussed.


It is generally accepted that, following primary infection, human cytomegalovirus (HCMV) establishes lifelong latency in CD34(+) progenitor cells and other derivative cells of the myeloid lineage. In this study, we show that the viral UL144 gene is expressed during latent infection in two cell types of the myeloid lineage, CD34(+) and CD14(+) monocytes, and that the UL144 protein is functional in latently infected monocytes. However, this latency-associated expression of UL144 occurs only in certain isolates of HCMV and depends on the presence of functional GATA-2 transcription factor binding sites in the UL144 promoter, in contrast to the viral latency-associated gene LUNA, which we also show is regulated by GATA-2 but expressed uniformly during latent infection independent of the virus isolate. Taken together, these data suggest that the HCMV latency-associated transcriptome may be virus isolate specific and dependent on the repertoire of transcription factor binding sites in the promoters of latency-associated genes.


Lowering low-density lipoprotein-cholesterol (LDL-C) is the primary target in the management of dyslipidemia in patients at high risk of cardiovascular disease. However, patients who have achieved LDL-C levels below the currently recommended targets may still experience cardiovascular events. This may result, in part, from elevated triglyceride (TG) levels and low levels of high-density lipoprotein-cholesterol (HDL-C). Low HDL-C and high TG levels are common and are recognized as independent risk factors for cardiovascular morbidity and mortality. Furthermore, atherogenic dyslipidemia, characterized by low levels of HDL-C, high TG, and small, dense LDL particles, is a typical phenotype of dyslipidemia in subjects with insulin resistance and metabolic syndrome. Therefore, to reduce further the risk of coronary heart disease (CHD), raising HDL-C and lowering TG may be the secondary therapeutic target for patients who achieve LDL-C levels below the currently recommended targets but are still at risk of CHD. However, whether increasing HDL-C levels alone reduces CHD has not yet been confirmed in large randomized clinical trials, and whether functional HDL is more important than HDL-C in reducing CHD remains controversial. Large CHD endpoint trials that include many patients with diabetes are underway to compare combination treatments with statin and niacin, fibrates, or cholesteryl ester transfer protein inhibitors with statin alone treatments. In this review, we discuss the rationale and importance of increasing HDL-C levels with and without lowering TG levels in the treatment and prevention of cardiovascular events.


AIMS/HYPOTHESIS We assessed the association between congenital malformations and maternal hyperglycemia in pregnant women with pregestational (type 1 or type 2) diabetes and investigated if the rate of congenital malformations was similar in women with near-normal glycemic control compared to the background population. We also assessed the association between congenital malformations and maternal hyperglycemia in pregnant women with pregestational diabetes with special focus on women with near-normal HbA1c in early pregnancy. MATERIALS AND METHODS This is a literature review based on an electronic literature search of the databases PubMed, Cochrane, Embase and Web of Science conducted in July 2017 using the search terms diabetes, pregnancy, HbA1c or glycemic control and congenital anomaly or congenital anomaly. We included original papers in English published after 1997 with data on congenital malformations and HbA1c in at least 250 women with pregestational diabetes. RESULTS Nine papers with in total 6225 women with type 1 diabetes and 2334 women with type 2 diabetes were included. The prevalence of congenital malformations was 6.4% in women with type 1 diabetes and 4.3% in women with type 2 diabetes and for the combined group of women with pregestational diabetes, the relative risk compared to the background population was 3.2. In women with HbA1c < 53 mmol/mol (7.0%) in early pregnancy or HbA1c 53-64 mmol/mol (7.0-8.0%) the prevalence of congenital malformations was 4.3 and 3.7%, respectively, with a relative risk of 2.2 and 1.9, respectively. CONCLUSIONS In pregnant women with pregestational diabetes the prevalence of congenital abnormalities was threefold higher in women with pregestational diabetes compared to the background population. However, HbA1c below 53 mmol/mol (7.0%) in early pregnancy was also associated with a two times increased risk of congenital malformations compared to the background population.


We show that NF-kappaB and transcriptional targets are activated in liver by obesity and high-fat diet (HFD). We have matched this state of chronic, subacute 'inflammation' by low-level activation of NF-kappaB in the liver of transgenic mice, designated LIKK, by selectively expressing constitutively active IKK-b in hepatocytes. These mice exhibit a type 2 diabetes phenotype, characterized by hyperglycemia, profound hepatic insulin resistance, and moderate systemic insulin resistance, including effects in muscle. The hepatic production of proinflammatory cytokines, including IL-6, IL-1beta and TNF-alpha, was increased in LIKK mice to a similar extent as induced by HFD in in wild-type mice. Parallel increases were observed in cytokine signaling in liver and mucscle of LIKK mice. Insulin resistance was improved by systemic neutralization of IL-6 or salicylate inhibition of IKK-beta. Hepatic expression of the IkappaBalpha superrepressor (LISR) reversed the phenotype of both LIKK mice and wild-type mice fed an HFD. These findings indicate that lipid accumulation in the liver leads to subacute hepatic 'inflammation' through NF-kappaB activation and downstream cytokine production. This causes insulin resistance both locally in liver and systemically.


PURPOSE OF REVIEW The aim of this study is to highlight some recent uses of serum metabolomics in human and animal studies. The main themes are the importance of understanding the underlying variation in human metabolism and the use of serum metabolomics in disease profiling. RECENT FINDINGS Several studies have attempted to use serum metabolomics to develop noninvasive biomarkers of disease and/or track the consequences of nutritional and genetic interventions. Many advances have been made with common changes being identified in ageing, the menopause and cancer but several problems of interpretation have emerged from these studies. These include the small sample sizes in most human studies and the differences between human and rodent metabolomes. However, a metabolic screen of over 1000 'healthy' humans (the Humsermet project) has highlighted many variables that may be used to refine the interpretation and design of previous and future human studies alike, in addition to data mining. SUMMARY Some common serum metabolome alterations have been identified but many inconsistencies remain. The construction of a human serum metabolome database should be informative in the design of future human and animal model studies.


G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation.


Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group (PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses nonskin lineages and HF differentiation. In TA progeny, nonskin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks, and HF-lineage regulators lose them. Interestingly, genes poised in embryonic stem cells, active in HF-SCs, and PcG-repressed in TA progeny encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our findings explain how HF-SCs cycle through quiescent and activated states without losing stemness and define roles for PcG-mediated repression in governing a fate switch irreversibly.


BACKGROUND Patients with type 2 diabetes have a 40% increased risk of bladder cancer. Thiazolidinediones, especially pioglitazone, may increase the risk. We conducted a systematic review and meta-analysis to evaluate the risk of bladder cancer among adults with type 2 diabetes taking thiazolidinediones. METHODS We searched key biomedical databases (including MEDLINE, Embase and Scopus) and sources of grey literature from inception through March 2012 for published and unpublished studies, without language restrictions. We included randomized controlled trials (RCTs), cohort studies and case-control studies that reported incident bladder cancer among people with type 2 diabetes who ever (v. never) were exposed to pioglitazone (main outcome), rosiglitazone or any thiazolidinedione. RESULTS Of the 1787 studies identified, we selected 4 RCTs, 5 cohort studies and 1 case-control study. The total number of patients was 2,657,365, of whom 3643 had newly diagnosed bladder cancer, for an overall incidence of 53.1 per 100,000 person-years. The one RCT that reported on pioglitazone use found no significant association with bladder cancer (risk ratio [RR] 2.36, 95% confidence interval [CI] 0.91-6.13). The cohort studies of thiazolidinediones (pooled RR 1.15, 95% CI 1.04-1.26; I(2) = 0%) and of pioglitazone specifically (pooled RR 1.22, 95% CI 1.07-1.39; I(2) = 0%) showed significant associations with bladder cancer. No significant association with bladder cancer was observed in the two RCTs that evaluated rosiglitazone use (pooled RR 0.87, 95% CI 0.34-2.23; I(2) = 0%). INTERPRETATION The limited evidence available supports the hypothesis that thiazolidinediones, particularly pioglitazone, are associated with an increased risk of bladder cancer among adults with type 2 diabetes.


The RIG-I-like receptors (RLRs) RIG-I, MDA5, and LGP2 play a major role in pathogen sensing of RNA virus infection to initiate and modulate antiviral immunity. The RLRs detect viral RNA ligands or processed self RNA in the cytoplasm to trigger innate immunity and inflammation and to impart gene expression that serves to control infection. Importantly, RLRs cooperate in signaling crosstalk networks with Toll-like receptors and other factors to impart innate immunity and to modulate the adaptive immune response. RLR regulation occurs at a variety of levels ranging from autoregulation to ligand and cofactor interactions and posttranslational modifications. Abberant RLR signaling or dysregulation of RLR expression is now implicated in the development of autoimmune diseases. Understanding the processes of RLR signaling and response will provide insights to guide RLR-targeted therapeutics for antiviral and immune-modifying applications.


Exposure to IR has been shown to induce the formation of senescence markers, a phenotype that coincides with lifelong delayed repair and regeneration of irradiated tissues. We hypothesized that IR-induced senescence markers could persist long-term in vivo, possibly contributing to the permanent reduction in tissue functionality. Here, we show that mouse tissues exposed to a sublethal dose of IR display persistent (up to 45 weeks, the maximum time analyzed) DNA damage foci and increased p16(INK4a) expression, two hallmarks of cellular senescence and aging. BrdU-labeling experiments revealed that IR-induced damaged cells are preferentially eliminated, at least partially, in a tissue-dependent manner. Unexpectedly, the accumulation of damaged cells was found to occur independent from the DNA damage response modulator p53, and from an intact immune system, as their levels were similar in wild-type and Rag2(-/-) gammaC(-/-) mice, the latter being deficient in T, B, and NK cells. Together, our results provide compelling evidence that exposure to IR induces long-term expression of senescence markers in vivo, an effect that may contribute to the reduced tissue functionality observed in cancer survivors.


Head development in vertebrates involves a complex series of molecular and morphogenetic events that generate a coordinated pattern of cartilages, bones and nerves, and result in species-specific craniofacial morphologies. A specialized cell type of neural origin, the neural crest, is central to this process, as it provides the main source of craniofacial mesenchyme. The degree of patterning information that is intrinsic to the neural crest has been recently debated, and new advances have underscored the influence of environmental signalling on the transcriptional readout that coordinates craniofacial morphogenesis in space and time.


We have previously demonstrated that, following acquisition of endocrine resistance, breast cancer cells display an altered growth rate together with increased aggressive behaviour in vitro. Since dysfunctional cell-cell adhesive interactions can promote an aggressive phenotype, we investigated the integrity of this protein complex in our breast cancer model of tamoxifen resistance. In culture, tamoxifen-resistant MCF7 (TamR) cells grew as loosely packed colonies with loss of cell-cell junctions and demonstrated altered morphology characteristic of cells undergoing epithelial-to-mesenchymal transition (EMT). Neutralising E-cadherin function promoted the invasion and inhibited the aggregation of endocrine-sensitive MCF7 cells, whilst having little effect on the behaviour of TamR cells. Additionally, TamR cells had increased levels of tyrosine-phosphorylated beta-catenin, whilst serine/threonine-phosphorylated beta-catenin was decreased. These cells also displayed loss of association between beta-catenin and E-cadherin, increased cytoplasmic and nuclear beta-catenin and elevated transcription of beta-catenin target genes known to be involved in tumour progression and EMT. Inhibition of EGFR kinase activity in TamR cells reduced beta-catenin tyrosine phosphorylation, increased beta-catenin-E-cadherin association and promoted cell-cell adhesion. In such treated cells, the association of beta-catenin with Lef-1 and the transcription of c-myc, cyclin-D1, CD44 and COX-2 were also reduced. These results suggest that homotypic adhesion in tamoxifen-resistant breast cancer cells is dysfunctional due to EGFR-driven modulation of the phosphorylation status of beta-catenin and may contribute to an enhanced aggressive phenotype and transition towards a mesenchymal phenotype in vitro.


Patients with idiopathic pulmonary fibrosis (IPF) have a higher incidence             of lung cancer. The role of Toll-like receptors (TLRs), a key component of the             innate immunity, in interstitial lung diseases (ILDs) and lung cancer pathogenesis             is not clarified. TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 mRNA expression was quantitatively             measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR)             in bronchoalveolar lavage fluid (BALF) of 16 IPF patients, 16 non-small cell lung             cancer (NSCLC) patients and 9 control subjects. TLR2, TLR3, TLR4 and TLR9 protein             expression was assessed on BALF T-lymphocytes using flow cytometry. TLR3 mRNA             expression was significantly higher in NSCLC compared to IPF (p=0.023) and controls             (p=0.001). TLR7 mRNA expression levels were significantly higher in both NSCLC             and IPF groups compared to controls (p=0.029, p=0.009). TLR9 expression at the             mRNA level was significantly higher in both NSCLC and IPF groups compared to controls             (p=0.01, p=0.001). Finally, TLR2 mRNA expression was significantly higher in IPF             patients compared to controls (p=0.042). Flow cytometry revealed decreased TLR3             and TLR9 expression in IPF patients compared to the NSCLC group (p=0.02, p=0.014)             and decreased TLR9 expression in IPF compared with the controls (p=0.04). TLR2             protein expression was significantly higher in IPF patients compared to NSCLC             (p=0.04). Increased expression of endosomal TLRs in NSCLC patients and elevated             expression of TLR2 in pulmonary fibrosis are the main results of this study. These             results do not provide support for a common TLR pathway hypothesis between NSCLC             and IPF.


Progerias are rare genetic diseases characterized by premature aging. Several progeroid disorders are caused by mutations that lead to the accumulation of a lipid-modified (farnesylated) form of prelamin A, a protein that contributes to the structural scaffolding for the cell nucleus. In progeria, the accumulation of farnesyl-prelamin A disrupts this scaffolding, leading to misshapen nuclei. Previous studies have shown that farnesyltransferase inhibitors (FTIs) reverse this cellular abnormality. We tested the efficacy of an FTI (ABT-100) in Zmpste24-deficient mice, a mouse model of progeria. The FTI-treated mice exhibited improved body weight, grip strength, bone integrity, and percent survival at 20 weeks of age. These results suggest that FTIs may have beneficial effects in humans with progeria.


BACKGROUND Patients with human immunodeficiency virus (HIV) infection and tuberculosis have an increased risk of death, treatment failure, and relapse. METHODS A systematic review and meta-analysis of randomized, controlled trials and cohort studies was conducted to evaluate the impact of duration and dosing schedule of rifamycin and use of antiretroviral therapy in the treatment of active tuberculosis in HIV-positive patients. In included studies, the initial tuberculosis diagnosis, failure, and/or relapse were microbiologically confirmed, and patients received standardized rifampin- or rifabutin-containing regimens. Pooled cumulative incidence of treatment failure, death during treatment, and relapse were calculated using random-effects models. Multivariable meta-regression was performed using negative binomial regression. RESULTS After screening 5158 citations, 6 randomized trials and 21 cohort studies were included. Relapse was more common with regimens using 2 months rifamycin (adjusted risk ratio, 3.6; 95% confidence interval, 1.1-11.7) than with regimens using rifamycin for at least 8 months. Compared with daily therapy in the initial phase (n=3352 patients from 35 study arms), thrice-weekly therapy (n=211 patients from 5 study arms) was associated with higher rates of failure (adjusted risk ratio, 4.0; 95% confidence interval, 1.5-10.4) and relapse [adjusted risk ratio, 4.8; 95% confidence interval, 1.8-12.8). There were trends toward higher relapse rates if rifamycins were used for only 6 months, compared with > or =8 months, or if antiretroviral therapy was not used. CONCLUSIONS This review raises serious concerns regarding current recommendations for treatment of HIV-tuberculosis coinfection. The data suggest that at least 8 months duration of rifamycin therapy, initial daily dosing, and concurrent antiretroviral therapy might be associated with better outcomes, but adequately powered randomized trials are urgently needed to confirm this.


Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.


The loss of the ability to deaminate l-serine severely impairs growth and cell division in Escherichia coli K-12. A strain from which the three genes (sdaA, sdaB, tdcG) coding for this organism's three l-serine deaminases had been deleted grows well in glucose minimal medium but, on subculture into minimal medium with glucose and casamino acids, it makes very large, abnormally shaped cells, many of which lyse. When inoculated into Luria-Bertani (LB) broth with or without glucose, it makes very long filaments. Provision of S-adenosylmethionine restores cell division in LB broth with glucose, and repairs much of the difficulty in growth in medium with casamino acids. We suggest that replication of E. coli is regulated by methylation, that an unusually high intracellular l-serine concentration, in the presence of other amino acids, starves the cell for S-adenosylmethionine and that it is the absence of S-adenosylmethionine and/or of C1-tetrahydrofolate derivatives that prevents normal cell division.