Question: The NF-κB essential modulator gene induces intestinal inflammation by promoting receptor-interacting protein (RIP) kinase 1 kinase activity-mediated epithelial cell death.

NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions. Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor κB (NF-κB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-κB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.

NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells.. The phagocyte NADPH oxidase (NOX2) is critical for the bactericidal activity of phagocytic cells and plays a major role in innate immunity. We showed recently that NOX2 activity in mouse dendritic cells (DCs) prevents acidification of phagosomes, promoting antigen cross-presentation. In order to investigate the role of NOX2 in the regulation of the phagosomal pH in human DCs, we analyzed the production of reactive oxygen species (ROS) and the phagosomal/endosomal pH in monocyte-derived DCs and macrophages (M(diameter)s) from healthy donors or patients with chronic granulomatous disease (CGD). As expected, we found that human M(diameter)s acidify their phagosomes more efficiently than human DCs. Accordingly, the expression of the vacuolar proton ATPase (V-H(+)-ATPase) was higher in M(diameter)s than in DCs. Phagosomal ROS production, however, was also higher in M(diameter)s than in DCs, due to higher levels of gp91phox expression and recruitment to phagosomes. In contrast, in the absence of active NOX2, the phagosomal and endosomal pH decreased. Both in the presence of a NOX2 inhibitor and in DCs derived from patients with CGD, the cross-presentation of 2 model tumor antigens was impaired. We conclude that NOX2 activity participates in the regulation of the phagosomal and endosomal pH in human DCs, and is required for efficient antigen cross-presentation.


Question: One in two surgical randomized controlled trials are discontinued early.

Discontinuation and non-publication of surgical randomised controlled trials: observational study. OBJECTIVE To determine the rate of early discontinuation and non-publication of randomised controlled trials involving patients undergoing surgery. DESIGN Cross sectional observational study of registered and published trials. SETTING Randomised controlled trials of interventions in patients undergoing a surgical procedure. DATA SOURCES The ClinicalTrials.gov database was searched for interventional trials registered between January 2008 and December 2009 using the keyword "surgery". Recruitment status was extracted from the ClinicalTrials.gov database. A systematic search for studies published in peer reviewed journals was performed; if they were not found, results posted on the ClinicalTrials.gov results database were sought. Email queries were sent to trial investigators of discontinued and unpublished completed trials if no reason for the respective status was disclosed. MAIN OUTCOME MEASURES Trial discontinuation before completion and non-publication after completion. Logistic regression was used to determine the effect of funding source on publication status, with adjustment for intervention type and trial size. RESULTS Of 818 registered trials found using the keyword "surgery", 395 met the inclusion criteria. Of these, 21% (81/395) were discontinued early, most commonly owing to poor recruitment (44%, 36/81). The remaining 314 (79%) trials proceeded to completion, with a publication rate of 66% (208/314) at a median time of 4.9 (interquartile range 4.0-6.0) years from study completion to publication search. A further 6% (20/314) of studies presented results on ClinicalTrials.gov without a corresponding peer reviewed publication. Industry funding did not affect the rate of discontinuation (adjusted odds ratio 0.91, 95% confidence interval 0.54 to 1.55) but was associated with a lower odds of publication for completed trials (0.43, 0.26 to 0.72). Investigators' email addresses for trials with an uncertain fate were identified for 71.4% (10/14) of discontinued trials and 83% (101/122) of unpublished studies. Only 43% (6/14) and 20% (25/122) replies were received. Email responses for completed trials indicated 11 trials in press, five published studies (four in non-indexed peer reviewed journals), and nine trials remaining unpublished. CONCLUSIONS One in five surgical randomised controlled trials are discontinued early, one in three completed trials remain unpublished, and investigators of unpublished studies are frequently not contactable. This represents a waste of research resources and raises ethical concerns regarding hidden clinical data and futile participation by patients with its attendant risks. To promote future efficiency and transparency, changes are proposed to research governance frameworks to overcome these concerns.

Tricyclic antidepressants and headaches: systematic review and meta-analysis. OBJECTIVE To evaluate the efficacy and relative adverse effects of tricyclic antidepressants in the treatment of migraine, tension-type, and mixed headaches. DESIGN Meta-analysis. DATA SOURCES Medline, Embase, the Cochrane Trials Registry, and PsycLIT. Studies reviewed Randomised trials of adults receiving tricyclics as only treatment for a minimum of four weeks. DATA EXTRACTION Frequency of headaches (number of headache attacks for migraine and number of days with headache for tension-type headaches), intensity of headache, and headache index. RESULTS 37 studies met the inclusion criteria. Tricyclics significantly reduced the number of days with tension-type headache and number of headache attacks from migraine than placebo (average standardised mean difference -1.29, 95% confidence interval -2.18 to -0.39 and -0.70, -0.93 to -0.48) but not compared with selective serotonin reuptake inhibitors (-0.80, -2.63 to 0.02 and -0.20, -0.60 to 0.19). The effect of tricyclics increased with longer duration of treatment (β=-0.11, 95% confidence interval -0.63 to -0.15; P<0.0005). Tricyclics were also more likely to reduce the intensity of headaches by at least 50% than either placebo (tension-type: relative risk 1.41, 95% confidence interval 1.02 to 1.89; migraine: 1.80, 1.24 to 2.62) or selective serotonin reuptake inhibitors (1.73, 1.34 to 2.22 and 1.72, 1.15 to 2.55). Tricyclics were more likely to cause adverse effects than placebo (1.53, 95% confidence interval 1.11 to 2.12) and selective serotonin reuptake inhibitors (2.22, 1.52 to 3.32), including dry mouth (P<0.0005 for both), drowsiness (P<0.0005 for both), and weight gain (P<0.001 for both), but did not increase dropout rates (placebo: 1.22, 0.83 to 1.80, selective serotonin reuptake inhibitors: 1.16, 0.81 to 2.97). CONCLUSIONS Tricyclic antidepressants are effective in preventing migraine and tension-type headaches and are more effective than selective serotonin reuptake inhibitors, although with greater adverse effects. The effectiveness of tricyclics seems to increase over time.


Question: Hypothalamic glutamate neurotransmission is unrelated to energy balance.

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

KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer. BACKGROUND Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas. METHODS We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally. RESULTS Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity. CONCLUSION These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.


Question: British female students are bullied more than British male students.

Bullying in school: are short pupils at risk? Questionnaire study in a cohort.. Bullying is still prevalent in schools and is clearly stressful for victims. 1 2 It may also have undesirable consequences for bullies, with antisocial behaviour persisting into adulthood. Victims are generally reported to be weaker than the bullies. 2 3 This would suggest that very short pupils are more likely to be victims and less likely to be the aggressors. The Wessex growth study allowed us to examine the prevalence of bullying, as experienced or perpetrated by pupils of different heights. Ninety two short normal adolescents who had been below the third centile for height at school entry4 and 117 controls matched for age and sex completed a bullying questionnaire, derived from work by Whitney and Smith.5 There were no refusals or any significant differences in sex or social class between the groups. Mean age (range) was 14.7 (13.4-15.7) years. Mean height SD scores were: short pupils −1.90 (−3.53 to −0.01), controls 0.31 (−1.41 …

THE EMBO JOURNAL. Glutamate-gated ion channels (ionotropic glutamate receptors, iGluRs) sense the extracellular milieu via an extensive extracellular portion, comprised of two clamshell-shaped segments. The distal, N-terminal domain (NTD) has allosteric potential in NMDA-type iGluRs, which has not been ascribed to the analogous domain in AMPA receptors (AMPARs). In this study, we present new structural data uncovering dynamic properties of the GluA2 and GluA3 AMPAR NTDs. GluA3 features a zipped-open dimer interface with unconstrained lower clamshell lobes, reminiscent of metabotropic GluRs (mGluRs). The resulting labile interface supports interprotomer rotations, which can be transmitted to downstream receptor segments. Normal mode analysis reveals two dominant mechanisms of AMPAR NTD motion: intraprotomer clamshell motions and interprotomer counter-rotations, as well as accessible interconversion between AMPAR and mGluR conformations. In addition, we detect electron density for a potential ligand in the GluA2 interlobe cleft, which may trigger lobe motions. Together, these data support a dynamic role for the AMPAR NTDs, which widens the allosteric landscape of the receptor and could provide a novel target for ligand development.


Question: The proliferative capacity of neural progenitors differs across species.

2D and 3D Stem Cell Models of Primate Cortical Development Identify Species-Specific Differences in Progenitor Behavior Contributing to Brain Size.. Variation in cerebral cortex size and complexity is thought to contribute to differences in cognitive ability between humans and other animals. Here we compare cortical progenitor cell output in humans and three nonhuman primates using directed differentiation of pluripotent stem cells (PSCs) in adherent two-dimensional (2D) and organoid three-dimensional (3D) culture systems. Clonal lineage analysis showed that primate cortical progenitors proliferate for a protracted period of time, during which they generate early-born neurons, in contrast to rodents, where this expansion phase largely ceases before neurogenesis begins. The extent of this additional cortical progenitor expansion differs among primates, leading to differences in the number of neurons generated by each progenitor cell. We found that this mechanism for controlling cortical size is regulated cell autonomously in culture, suggesting that primate cerebral cortex size is regulated at least in part at the level of individual cortical progenitor cell clonal output.

PI3K and cancer: lessons, challenges and opportunities. The central role of phosphoinositide 3-kinase (PI3K) activation in tumour cell biology has prompted a sizeable effort to target PI3K and/or downstream kinases such as AKT and mammalian target of rapamycin (mTOR) in cancer. However, emerging clinical data show limited single-agent activity of inhibitors targeting PI3K, AKT or mTOR at tolerated doses. One exception is the response to PI3Kδ inhibitors in chronic lymphocytic leukaemia, where a combination of cell-intrinsic and -extrinsic activities drive efficacy. Here, we review key challenges and opportunities for the clinical development of inhibitors targeting the PI3K–AKT–mTOR pathway. Through a greater focus on patient selection, increased understanding of immune modulation and strategic application of rational combinations, it should be possible to realize the potential of this promising class of targeted anticancer agents.


Question: ATF4 is a general endoplasmic reticulum stress marker.

Wolfram syndrome 1 and adenylyl cyclase 8 interact at the plasma membrane to regulate insulin production and secretion. Endoplasmic reticulum (ER) stress causes pancreatic β-cell dysfunction and contributes to β-cell loss and the progression of type 2 diabetes. Wolfram syndrome 1 (WFS1) has been shown to be an important regulator of the ER stress signalling pathway; however, its role in β-cell function remains unclear. Here we provide evidence that WFS1 is essential for glucose- and glucagon-like peptide 1 (GLP-1)-stimulated cyclic AMP production and regulation of insulin biosynthesis and secretion. Stimulation with glucose causes WFS1 translocation from the ER to the plasma membrane, where it forms a complex with adenylyl cyclase 8 (AC8), an essential cAMP-generating enzyme in the β-cell that integrates glucose and GLP-1 signalling. ER stress and mutant WFS1 inhibit complex formation and activation of AC8, reducing cAMP synthesis and insulin secretion. These findings reveal that an ER-stress-related protein has a distinct role outside the ER regulating both insulin biosynthesis and secretion. The reduction of WFS1 protein on the plasma membrane during ER stress is a contributing factor for β-cell dysfunction and progression of type 2 diabetes.

Molecular mechanisms of invadopodium formation: the role of the N-WASP–Arp2/3 complex pathway and cofilin. Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP–Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.


Question: Ca2+ cycling controls whole-body energy homeostasis in beige fat.

UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis. Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or β3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

Prognostic Accuracy of the SOFA Score, SIRS Criteria, and qSOFA Score for In-Hospital Mortality Among Adults With Suspected Infection Admitted to the Intensive Care Unit. Importance The Sepsis-3 Criteria emphasized the value of a change of 2 or more points in the Sequential [Sepsis-related] Organ Failure Assessment (SOFA) score, introduced quick SOFA (qSOFA), and removed the systemic inflammatory response syndrome (SIRS) criteria from the sepsis definition. Objective Externally validate and assess the discriminatory capacities of an increase in SOFA score by 2 or more points, 2 or more SIRS criteria, or a qSOFA score of 2 or more points for outcomes among patients who are critically ill with suspected infection. Design, Setting, and Participants Retrospective cohort analysis of 184 875 patients with an infection-related primary admission diagnosis in 182 Australian and New Zealand intensive care units (ICUs) from 2000 through 2015. Exposures SOFA, qSOFA, and SIRS criteria applied to data collected within 24 hours of ICU admission. Main Outcomes and Measures The primary outcome was in-hospital mortality. In-hospital mortality or ICU length of stay (LOS) of 3 days or more was a composite secondary outcome. Discrimination was assessed using the area under the receiver operating characteristic curve (AUROC). Adjusted analyses were performed using a model of baseline risk determined using variables independent of the scoring systems. Results Among 184 875 patients (mean age, 62.9 years [SD, 17.4]; women, 82 540 [44.6%]; most common diagnosis bacterial pneumonia, 32 634 [17.7%]), a total of 34 578 patients (18.7%) died in the hospital, and 102 976 patients (55.7%) died or experienced an ICU LOS of 3 days or more. SOFA score increased by 2 or more points in 90.1%; 86.7% manifested 2 or more SIRS criteria, and 54.4% had a qSOFA score of 2 or more points. SOFA demonstrated significantly greater discrimination for in-hospital mortality (crude AUROC, 0.753 [99% CI, 0.750-0.757]) than SIRS criteria (crude AUROC, 0.589 [99% CI, 0.585-0.593]) or qSOFA (crude AUROC, 0.607 [99% CI, 0.603-0.611]). Incremental improvements were 0.164 (99% CI, 0.159-0.169) for SOFA vs SIRS criteria and 0.146 (99% CI, 0.142-0.151) for SOFA vs qSOFA (P <.001). SOFA (AUROC, 0.736 [99% CI, 0.733-0.739]) outperformed the other scores for the secondary end point (SIRS criteria: AUROC, 0.609 [99% CI, 0.606-0.612]; qSOFA: AUROC, 0.606 [99% CI, 0.602-0.609]). Incremental improvements were 0.127 (99% CI, 0.123-0.131) for SOFA vs SIRS criteria and 0.131 (99% CI, 0.127-0.134) for SOFA vs qSOFA (P <.001). Findings were consistent for both outcomes in multiple sensitivity analyses. Conclusions and Relevance Among adults with suspected infection admitted to an ICU, an increase in SOFA score of 2 or more had greater prognostic accuracy for in-hospital mortality than SIRS criteria or the qSOFA score. These findings suggest that SIRS criteria and qSOFA may have limited utility for predicting mortality in an ICU setting.


Question: 1 in 5 million in UK have abnormal PrP positivity.

Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey. OBJECTIVES To carry out a further survey of archived appendix samples to understand better the differences between existing estimates of the prevalence of subclinical infection with prions after the bovine spongiform encephalopathy epizootic and to see whether a broader birth cohort was affected, and to understand better the implications for the management of blood and blood products and for the handling of surgical instruments. DESIGN Irreversibly unlinked and anonymised large scale survey of archived appendix samples. SETTING Archived appendix samples from the pathology departments of 41 UK hospitals participating in the earlier survey, and additional hospitals in regions with lower levels of participation in that survey. SAMPLE 32,441 archived appendix samples fixed in formalin and embedded in paraffin and tested for the presence of abnormal prion protein (PrP). RESULTS Of the 32,441 appendix samples 16 were positive for abnormal PrP, indicating an overall prevalence of 493 per million population (95% confidence interval 282 to 801 per million). The prevalence in those born in 1941-60 (733 per million, 269 to 1596 per million) did not differ significantly from those born between 1961 and 1985 (412 per million, 198 to 758 per million) and was similar in both sexes and across the three broad geographical areas sampled. Genetic testing of the positive specimens for the genotype at PRNP codon 129 revealed a high proportion that were valine homozygous compared with the frequency in the normal population, and in stark contrast with confirmed clinical cases of vCJD, all of which were methionine homozygous at PRNP codon 129. CONCLUSIONS This study corroborates previous studies and suggests a high prevalence of infection with abnormal PrP, indicating vCJD carrier status in the population compared with the 177 vCJD cases to date. These findings have important implications for the management of blood and blood products and for the handling of surgical instruments.

Cell-cell adhesion and signalling.. Signalling pathways activated by Rho small GTPases have recently been identified that coordinate junction assembly, stability and function, as well as interactions of adhesive complexes with the underlying cortical cytoskeleton. Particularly exciting is the interplay between adherens junctions, activation of Rho proteins and the dynamics of microtubule, actin and intermediate filaments. This interplay has important implications for functional regulation of cell-cell adhesion, and points to a more integrated view of signalling processes.


Question: Cis-acting lncRNAs control the expression of genes that are positioned in the vicinity of their transcription sites.

Functional Consequences of Splicing of the Antisense Transcript COOLAIR on FLC Transcription. Antisense transcription is widespread in many genomes; however, how much is functional is hotly debated. We are investigating functionality of a set of long noncoding antisense transcripts, collectively called COOLAIR, produced at Arabidopsis FLOWERING LOCUS C (FLC). COOLAIR initiates just downstream of the major sense transcript poly(A) site and terminates either early or extends into the FLC promoter region. We now show that splicing of COOLAIR is functionally important. This was revealed through analysis of a hypomorphic mutation in the core spliceosome component PRP8. The prp8 mutation perturbs a cotranscriptional feedback mechanism linking COOLAIR processing to FLC gene body histone demethylation and reduced FLC transcription. The importance of COOLAIR splicing in this repression mechanism was confirmed by disrupting COOLAIR production and mutating the COOLAIR proximal splice acceptor site. Our findings suggest that altered splicing of a long noncoding transcript can quantitatively modulate gene expression through cotranscriptional coupling mechanisms.

Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development. BACKGROUND Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development. METHODS AND FINDINGS Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression. CONCLUSIONS HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies. Please see later in the article for the Editors' Summary.


Question: LSD1-positive promoters are associated with RNA polymerase II

Histone Methylation-Dependent Mechanisms Impose Ligand Dependency for Gene Activation by Nuclear Receptors. Nuclear receptors undergo ligand-dependent conformational changes that are required for corepressor-coactivator exchange, but whether there is an actual requirement for specific epigenetic landmarks to impose ligand dependency for gene activation remains unknown. Here we report an unexpected and general strategy that is based on the requirement for specific cohorts of inhibitory histone methyltransferases (HMTs) to impose gene-specific gatekeeper functions that prevent unliganded nuclear receptors and other classes of regulated transcription factors from binding to their target gene promoters and causing constitutive gene activation in the absence of stimulating signals. This strategy, based at least in part on an HMT-dependent inhibitory histone code, imposes a requirement for specific histone demethylases, including LSD1, to permit ligand- and signal-dependent activation of regulated gene expression. These events link an inhibitory methylation component of the histone code to a broadly used strategy that circumvents pathological constitutive gene induction by physiologically regulated transcription factors.

CCR2 and CXCR4 regulate peripheral blood monocyte pharmacodynamics and link to efficacy in experimental autoimmune encephalomyelitis. BACKGROUND CCR2 plays a key role in regulating monocyte trafficking to sites of inflammation and therefore has been the focus of much interest as a target for inflammatory disease. METHODS Here we examined the effects of CCR2 blockade with a potent small molecule antagonist to determine the pharmacodynamic consequences on the peripheral blood monocyte compartment in the context of acute and chronic inflammatory processes. RESULTS We demonstrate that CCR2 antagonism in vivo led to a rapid decrease in the number of circulating Ly6Chi monocytes and that this decrease was largely due to the CXCR4-dependent sequestration of these cells in the bone marrow, providing pharmacological evidence for a mechanism by which monocyte dynamics are regulated in vivo. CCR2 antagonism led to an accumulation of circulating CCL2 and CCL7 levels in the blood, indicating a role for CCR2 in regulating the levels of its ligands under homeostatic conditions. Finally, we show that the pharmacodynamic changes due to CCR2 antagonism were apparent after chronic dosing in mouse experimental autoimmune encephalomyelitis, a model in which CCR2 blockade demonstrated a dramatic reduction in disease severity, manifest in a reduced accumulation of monocytes and other cells in the CNS. CONCLUSION CCR2 antagonism in vivo has tractable pharmacodynamic effects that can be used to align target engagement with biologic effects on disease activity.


Question: Patients with common epithelial cancers  are more likely to have an emergency event as their first hospital admission if they live in resource-deprived areas.

Social variations in access to hospital care for patients with colorectal, breast, and lung cancer between 1999 and 2006: retrospective analysis of hospital episode statistics. OBJECTIVES To determine the extent to which type of hospital admission (emergency compared with elective) and surgical procedure varied by socioeconomic circumstances, age, sex, and year of admission for colorectal, breast, and lung cancer. DESIGN Repeated cross sectional study with data from individual patients, 1 April 1999 to 31 March 2006. SETTING Hospital episode statistics (HES) dataset. PARTICIPANTS 564 821 patients aged 50 and over admitted with a diagnosis of colorectal, breast, or lung cancer. MAIN OUTCOME MEASURES Proportion of patients admitted as emergencies, and the proportion receiving the recommended surgical treatment. RESULTS Patients from deprived areas, older people, and women were more likely to be admitted as emergencies. For example, the adjusted odds ratio for patients with breast cancer in the least compared with most deprived fifth of deprivation was 0.63 (95% confidence interval 0.60 to 0.66) and the adjusted odds ratio for patients with lung cancer aged 80-89 compared with those aged 50-59 was 3.13 (2.93 to 3.34). There were some improvements in disparities between age groups but not for patients living in deprived areas over time. Patients from deprived areas were less likely to receive preferred procedures for rectal, breast, and lung cancer. These findings did not improve with time. For example, 67.4% (3529/5237) of patients in the most deprived fifth of deprivation had anterior resection for rectal cancer compared with 75.5% (4497/5959) of patients in the least deprived fifth (1.34, 1.22 to 1.47). Over half (54.0%, 11 256/20 849) of patients in the most deprived fifth of deprivation had breast conserving surgery compared with 63.7% (18 445/28 960) of patients in the least deprived fifth (1.21, 1.16 to 1.26). Men were less likely than women to undergo anterior resection and lung cancer resection and older people were less likely to receive breast conserving surgery and lung cancer resection. For example, the adjusted odds ratio for lung cancer patients aged 80-89 compared with those aged 50-59 was 0.52 (0.46 to 0.59). Conclusions Despite the implementation of the NHS Cancer Plan, social factors still strongly influence access to and the provision of care.

Coming soon to a Wal-Mart near you. According to the Web site of the Association for Automatic Identification and Data Capture Technologies [http://www.aimglobal.org/technologies/rfid], "radio frequency identification (RFID) technology is an automatic way to collect product, place, and time or transaction data quickly and easily without human intervention or error. " With the ability to track everything from crates of disposable razors to individual peanut-butter jars on the store shelves, RFID technology offers the potential of "real-time supply chain visibility. " Promoters of RFID technology feel [C. Humer, 2003] that "RF tags are to this decade what the Internet was to the 1990's-a promise of radical change in the way business is done. " However, before the full potential of RFID technology can be realized, several hurdles need to be overcome: reliability, cost, lack of standards, and security. As these hurdles gradually diminish, Wal-Mart publicly embraces the technology.


Question: T cell receptor /CD3 microdomains are required to induce the immunologic synapse.

T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster.. T cell receptor (TCR) signaling is initiated and sustained in microclusters; however, it's not known whether signaling also occurs in the TCR-rich central supramolecular activation cluster (cSMAC). We showed that the cSMAC formed by fusion of microclusters contained more CD45 than microclusters and is a site enriched in lysobisphosphatidic acid, a lipid involved in sorting ubiquitinated membrane proteins for degradation. Calcium signaling via TCR was blocked within 2 min by anti-MHCp treatment and 1 min by latrunculin-A treatment. TCR-MHCp interactions in the cSMAC survived these perturbations for 10 min and hence were not sufficient to sustain signaling. TCR microclusters were also resistant to disruption by anti-MHCp and latrunculin-A treatments. We propose that TCR signaling is sustained by stabilized microclusters and is terminated in the cSMAC, a structure from which TCR are sorted for degradation. Our studies reveal a role for F-actin in TCR signaling beyond microcluster formation.

Rapid clearance of human papillomavirus and implications for clinical focus on persistent infections.. Health professionals and the public need to understand the natural history of human papillomavirus (HPV) infections of the cervix to best use the information provided by new molecular screening tests. We investigated outcomes of 800 carcinogenic HPV infections detected in 599 women at enrollment into a population-based cohort (Guanacaste, Costa Rica). For individual infections, we calculated cumulative proportions of three outcomes (viral clearance, persistence without cervical intraepithelial neoplasia grade 2 or worse [CIN2+], or persistence with new diagnosis of CIN2+) at successive 6-month time points for the first 30 months of follow-up. Cervical specimens were tested for carcinogenic HPV genotypes using an L1 degenerate-primer polymerase chain reaction method. Infections typically cleared rapidly, with 67% (95% confidence interval [CI] = 63% to 70%) clearing by 12 months. However, among infections that persisted at least 12 months, the risk of CIN2+ diagnosis by 30 months was 21% (95% CI = 15% to 28%). The risk of CIN2+ diagnosis was highest among women younger than 30 years with HPV-16 infections that persisted for at least 12 months (53%; 95% CI = 29% to 76%). These findings suggest that the medical community should emphasize persistence of cervical HPV infection, not single-time detection of HPV, in management strategies and health messages.


Question: mcm 5 s 2 U is required for proper decoding of NNR codons in eukaryotes.

Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions. The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm(5)s(2)U by largely fixing the C3'-endo ribose puckering, ensuring stable and accurate codon-anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm(5)s(2)U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

Neutrophil extracellular traps capture and kill Candida albicans yeast and hyphal forms.. Neutrophils phagocytose and kill microbes upon phagolysosomal fusion. Recently we found that activated neutrophils form extracellular fibres that consist of granule proteins and chromatin. These neutrophil extracellular traps (NETs) degrade virulence factors and kill Gram positive and negative bacteria. Here we show for the first time that Candida albicans, a eukaryotic pathogen, induces NET-formation and is susceptible to NET-mediated killing. C. albicans is the predominant aetiologic agent of fungal infections in humans, particularly in immunocompromised hosts. One major virulence trait of C. albicans is its ability to reversibly switch from singular budding cells to filamentous hyphae. We demonstrate that NETs kill both yeast-form and hyphal cells, and that granule components mediate fungal killing. Taken together our data indicate that neutrophils trap and kill ascomycetous yeasts by forming NETs.


Question: Genetic deficiency of mast cells leads to decreased weight gain in a mouse model of diet-induced obesity.

Deficiency and pharmacological stabilization of mast cells reduce diet-induced obesity and diabetes in mice. Although mast cell functions have classically been related to allergic responses, recent studies indicate that these cells contribute to other common diseases such as multiple sclerosis, rheumatoid arthritis, atherosclerosis, aortic aneurysm and cancer. This study presents evidence that mast cells also contribute to diet-induced obesity and diabetes. For example, white adipose tissue (WAT) from obese humans and mice contain more mast cells than WAT from their lean counterparts. Furthermore, in the context of mice on a Western diet, genetically induced deficiency of mast cells, or their pharmacological stabilization, reduces body weight gain and levels of inflammatory cytokines, chemokines and proteases in serum and WAT, in concert with improved glucose homeostasis and energy expenditure. Mechanistic studies reveal that mast cells contribute to WAT and muscle angiogenesis and associated cell apoptosis and cathepsin activity. Adoptive transfer experiments of cytokine-deficient mast cells show that these cells, by producing interleukin-6 (IL-6) and interferon-gamma (IFN-gamma), contribute to mouse adipose tissue cysteine protease cathepsin expression, apoptosis and angiogenesis, thereby promoting diet-induced obesity and glucose intolerance. Our results showing reduced obesity and diabetes in mice treated with clinically available mast cell-stabilizing agents suggest the potential of developing new therapies for these common human metabolic disorders.

Partitioning the symptoms of hypoglycaemia using multi-sample confirmatory factor analysis. The allocation of hypoglycaemic symptoms to autonomie or neuroglycopenic groups tends to occur on an a priori basis. In view of the practical need for clear symptom markers of hypoglycaemia more scientific approaches must be pursued. Substantial evidence is presented from two large scale studies we performed which support a three factor model of hypoglycaemic symptomatology, based on the statistical associations discovered among symptoms reported by diabetic patients. Study 1 involved 295 insulin-treated outpatients and found that 11 key hypoglycaemic symptoms segregated into three clear factors: autonomie (sweating, palpitation, shaking and hunger) neuroglycopenic (confusion, drowsiness, odd behaviour, speech difficulty and incoordination), and malaise (nausea and headache). The three factors were validated on a separate group of 303 insulin-treated diabetic out-patients. Confirmatory factor analyses showed that the three factor model was the optimal model for explaining symptom covariance in each group. A multi-sample confirmatory factor analysis tested the rigorous assumptions that the relative loadings of symptoms on factors across groups were equal, and that the residual variance for each symptom was identical across groups. These assumptions were successful, indicating that the three factor model was replicated in detail across these two large samples. It is suggested that the results indicate valid groupings of symptoms that may be used in future research and in clinical practice.


Question: ScPif1p has reduced binding ability to G-rich ssDNA compared to non-G-rich ssDNA.

Pif1 family helicases suppress genome instability at G-quadruplex motifs. The Saccharomyces cerevisiae Pif1 helicase is the prototypical member of the Pif1 DNA helicase family, which is conserved from bacteria to humans. Here we show that exceptionally potent G-quadruplex unwinding is conserved among Pif1 helicases. Moreover, Pif1 helicases from organisms separated by more than 3 billion years of evolution suppressed DNA damage at G-quadruplex motifs in yeast. The G-quadruplex-induced damage generated in the absence of Pif1 helicases led to new genetic and epigenetic changes. Furthermore, when expressed in yeast, human PIF1 suppressed both G-quadruplex-associated DNA damage and telomere lengthening.

Magnetic resonance imaging for volume estimation of large multinodular goitres: a comparison with scintigraphy.. As a result of increasing interest in non-surgical treatment for the reduction of goitre size the use of magnetic resonance (MR) imaging for volume estimation of large multinodular goitres was evaluated in 20 patients (three males and 17 females; age 61 +/- 21 years) with a multinodular goitre larger than 100 ml. In addition, MR measurements were compared with scintigraphic (SC) volume estimations. Intraobserver coefficient of variation (CV) of MR measurements was 2.2 +/- 2.0% (Observer 1) and interobserver CV 4.1 +/- 2.2% (Observers 1 and 2). In all 20 patients signs of mechanical complications were shown on MR images. For SC measurements intraobserver CV was 7.5 +/- 5.7% (Observer 3) and 5.4 +/- 5.1% (Observer 4). Interobserver CV was 10.1 +/- 6.1%. The correlation between measurements with both methods was not strong (r = 0.665) and the resulting CV was 17.3 +/- 14.2%. Underestimation of SC volumes could not be explained by the presence of cysts on the surface of the thyroid. It is concluded that MR imaging can be used for in vivo thyroid volume estimation in large multinodular goitres. The high precision of MR measurements makes this technique potentially useful for the evaluation of thyroid growth and non-surgical treatment for reducing goitre size. Scintigraphic volume measurements do not suffice for this purpose. An additional advantage of MR imaging is the detailed anatomical information it provides with regard to mechanical complications of large goitres.


Question: In domesticated populations of Saccharomyces cerevisiae, segmental aneuploidy occurs infrequently.

Domestication and Divergence of Saccharomyces cerevisiae Beer Yeasts. Whereas domestication of livestock, pets, and crops is well documented, it is still unclear to what extent microbes associated with the production of food have also undergone human selection and where the plethora of industrial strains originates from. Here, we present the genomes and phenomes of 157 industrial Saccharomyces cerevisiae yeasts. Our analyses reveal that today's industrial yeasts can be divided into five sublineages that are genetically and phenotypically separated from wild strains and originate from only a few ancestors through complex patterns of domestication and local divergence. Large-scale phenotyping and genome analysis further show strong industry-specific selection for stress tolerance, sugar utilization, and flavor production, while the sexual cycle and other phenotypes related to survival in nature show decay, particularly in beer yeasts. Together, these results shed light on the origins, evolutionary history, and phenotypic diversity of industrial yeasts and provide a resource for further selection of superior strains. PAPERCLIP.

Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis.. Mononuclear phagocytes, including monocytes, macrophages, and dendritic cells, contribute to tissue integrity as well as to innate and adaptive immune defense. Emerging evidence for labor division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organization of this cellular network are not well defined. Here we report a fate-mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX(3)CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue-resident macrophage populations, including liver Kupffer cells and lung alveolar, splenic, and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that short-lived Ly6C(+) monocytes constitute obligatory steady-state precursors of blood-resident Ly6C(-) cells and that the abundance of Ly6C(+) blood monocytes dynamically controls the circulation lifespan of their progeny.


Question: Ribosomal protein (RP) expression is controlled in part by stress-activated regulators.

Pathway connectivity and signaling coordination in the yeast stress-activated signaling network. Stressed cells coordinate a multi-faceted response spanning many levels of physiology. Yet knowledge of the complete stress-activated regulatory network as well as design principles for signal integration remains incomplete. We developed an experimental and computational approach to integrate available protein interaction data with gene fitness contributions, mutant transcriptome profiles, and phospho-proteome changes in cells responding to salt stress, to infer the salt-responsive signaling network in yeast. The inferred subnetwork presented many novel predictions by implicating new regulators, uncovering unrecognized crosstalk between known pathways, and pointing to previously unknown 'hubs' of signal integration. We exploited these predictions to show that Cdc14 phosphatase is a central hub in the network and that modification of RNA polymerase II coordinates induction of stress-defense genes with reduction of growth-related transcripts. We find that the orthologous human network is enriched for cancer-causing genes, underscoring the importance of the subnetwork's predictions in understanding stress biology.

Ligand-target interactions: what can we learn from NMR?. The conformation of the ligand in complex with a macromolecular target can be studied by nuclear magnetic resonance (NMR) in solution for both tightly and weakly forming complexes. In the weak binding regime (k(off) > 10(4) Hz), the structure of the bound ligand is accessible also for very large complexes (>100 kDa), which are not amenable to NMR studies in the tight binding regime. Here I review the state-of-the-art NMR methodology used for screening ligands and for the structural investigation of bound ligand conformations, in both tight and weak binding regimes. The advantages and disadvantages of each approach are critically described. The NMR methodology used to investigate transiently forming complexes has expanded considerably in the past few years, opening new possibilities for a detailed description of ligand-target interactions. Novel methods for the determination of the bound ligand conformation, in particular transferred cross-correlated relaxation, are thoroughly reviewed, and their advantages with respect to established methodology are discussed, using the epothilone-tubulin complex as a primary example.


Question: Cellular differentiation of embryonic stem cells is accompanied by changes in replication timing, transcription and nuclear localization.

Global Reorganization of Replication Domains During Embryonic Stem Cell Differentiation. DNA replication in mammals is regulated via the coordinate firing of clusters of replicons that duplicate megabase-sized chromosome segments at specific times during S-phase. Cytogenetic studies show that these "replicon clusters" coalesce as subchromosomal units that persist through multiple cell generations, but the molecular boundaries of such units have remained elusive. Moreover, the extent to which changes in replication timing occur during differentiation and their relationship to transcription changes has not been rigorously investigated. We have constructed high-resolution replication-timing profiles in mouse embryonic stem cells (mESCs) before and after differentiation to neural precursor cells. We demonstrate that chromosomes can be segmented into multimegabase domains of coordinate replication, which we call "replication domains," separated by transition regions whose replication kinetics are consistent with large originless segments. The molecular boundaries of replication domains are remarkably well conserved between distantly related ESC lines and induced pluripotent stem cells. Unexpectedly, ESC differentiation was accompanied by the consolidation of smaller differentially replicating domains into larger coordinately replicated units whose replication time was more aligned to isochore GC content and the density of LINE-1 transposable elements, but not gene density. Replication-timing changes were coordinated with transcription changes for weak promoters more than strong promoters, and were accompanied by rearrangements in subnuclear position. We conclude that replication profiles are cell-type specific, and changes in these profiles reveal chromosome segments that undergo large changes in organization during differentiation. Moreover, smaller replication domains and a higher density of timing transition regions that interrupt isochore replication timing define a novel characteristic of the pluripotent state.

Critical role of CD28 in protective immunity against Salmonella typhimurium.. Efficient T cell activation requires both TCR signals and costimulatory signals. CD28 is one of the molecules that provide costimulatory signals for T cells. We used mice deficient in CD28 expression (CD28-/- mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever. CD28-/- mice were highly susceptible to infection with wild-type S. typhimurium and even failed to control infection with attenuated aroA- S. typhimurium. More detailed analysis revealed that CD28-/- animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-gamma. Thus, CD28 cosignaling is crucial for immunity against S. typhimurium. To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen.


Question: Upon viral challenge, influenza-specific memory CD4+ T cells greatly diminish the early production of inflammatory chemokines in the lung.

Memory CD4+ T cells induce innate responses independently of pathogen. Inflammation induced by recognition of pathogen-associated molecular patterns markedly affects subsequent adaptive responses. We asked whether the adaptive immune system can also affect the character and magnitude of innate inflammatory responses. We found that the response of memory, but not naive, CD4+ T cells enhances production of multiple innate inflammatory cytokines and chemokines (IICs) in the lung and that, during influenza infection, this leads to early control of virus. Memory CD4+ T cell–induced IICs and viral control require cognate antigen recognition and are optimal when memory cells are either T helper type 1 (TH1) or TH17 polarized but are independent of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) production and do not require activation of conserved pathogen recognition pathways. This represents a previously undescribed mechanism by which memory CD4+ T cells induce an early innate response that enhances immune protection against pathogens.

Aerobic capacity in black adolescent girls.. Our purpose was to determine the aerobic capacities (VO2max) of a group of black female adolescents (age = 11.4-15.8 years) randomly chosen from a single urban school. Of 91 girls selected, 64 performed an incremental treadmill running test to volitional exhaustion and achieved VO2max as determined from expired gas measures. Other measures included height (m), weight (kg), and calf and triceps skinfolds (for % fat estimates). Girls were also asked whether they had achieved menarche. VO2max averaged 37.3 +/- 6.2 ml.kg-1 x min-1 and was significantly correlated (r[62]) with height (-.32, p < .01), body mass index (-.63, p < .001), and % fat (-.65, p < .001) but not with age (-.16, p > .10). Postmenarchal girls were significantly taller and older than premenarchal girls. Contrary to previous studies, the girls' VO2max values were not related to biological age. Our subjects' aerobic capacity values averaged 14% less than those of nonblack U.S. female adolescents previously reported in the literature. This difference in VO2max was primarily a function of body weight. Study implications support the possibility that overweight in adult black women may originate prior to or during early adolescence. Future longitudinal studies should be designed to investigate the effects of aerobic fitness on cardiovascular risk factor reduction in black adolescent girls.


Question: RAD52 is involved in break-induced DNA replication (BIR).

Alternative Lengthening of Telomeres Mediated by Mitotic DNA Synthesis Engages Break-Induced Replication Processes.. Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. By analyzing telomerase-positive cells and their human TERC knockout-derived ALT human cell lines, we show that ALT cells harbor more fragile telomeres representing telomere replication problems. ALT-associated replication defects trigger mitotic DNA synthesis (MiDAS) at telomeres in a RAD52-dependent, but RAD51-independent, manner. Telomeric MiDAS is a conservative DNA synthesis process, potentially mediated by break-induced replication, similar to type II ALT survivors in Saccharomyces cerevisiae Replication stresses induced by ectopic oncogenic expression of cyclin E, G-quadruplexes, or R-loop formation facilitate the ALT pathway and lead to telomere clustering, a hallmark of ALT cancers. The TIMELESS/TIPIN complex suppresses telomere clustering and telomeric MiDAS, whereas the SMC5/6 complex promotes them. In summary, ALT cells exhibit more telomere replication defects that result in persistent DNA damage responses at telomeres, leading to the engagement of telomeric MiDAS (spontaneous mitotic telomere synthesis) that is triggered by DNA replication stress, a potential driver of genomic duplications in cancer.

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair. Cadherin-mediated cell-cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell-cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell-cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell-cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell-cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.


Question: Human embryonic stem cells give rise to cell types from all three embryonic germ layers.

Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst.. Somatic cell nuclear transfer (SCNT) technology has recently been used to generate animals with a common genetic composition. In this study, we report the derivation of a pluripotent embryonic stem (ES) cell line (SCNT-hES-1) from a cloned human blastocyst. The SCNT-hES-1 cells displayed typical ES cell morphology and cell surface markers and were capable of differentiating into embryoid bodies in vitro and of forming teratomas in vivo containing cell derivatives from all three embryonic germ layers in severe combined immunodeficient mice. After continuous proliferation for more than 70 passages, SCNT-hES-1 cells maintained normal karyotypes and were genetically identical to the somatic nuclear donor cells. Although we cannot completely exclude the possibility that the cells had a parthenogenetic origin, imprinting analyses support a SCNT origin of the derived human ES cells.

Effect of exercise-induced ischemia on QT interval dispersion.. An increased spatial dispersion of ventricular repolarization duration (QT dispersion) is associated with an increased vulnerability to arrhythmias. This study was designed to examine the effect of exercise on QT dispersion in ischemic heart disease (IHD). QT dispersion, corrected QT dispersion, and percentage change in uncorrected and corrected QT dispersion between rest and peak exercise were examined in 14 members of a control group, 17 patients with IHD, and 14 patients with IHD who were receiving beta-blockers (IHD-B). All subjects had undergone a standard Bruce protocol exercise test, and QT intervals were measured at rest and peak exercise with a digitizing tablet interfaced to a personal computer. QT dispersion at rest was markedly increased in the IHD group compared with that in the control and IHD-B groups, respectively (corrected QT dispersion in milliseconds), 74 +/- 7, 40 +/- 4, 49 +/- 5, p < 0.03). The corrected QT dispersion at peak exercise was greater in the IHD group compared with that in the control group (57 +/- 5 vs 26 +/- 3 msec, p < 0.03). The percentage change in QT dispersion with exercise was significantly higher in the IHD group (52% +/- 5%) compared with that in both the control group (28% +/- 4%, p < 0.002) and the IHD-B group (30% +/- 3%, p < 0.01). A larger mean QT dispersion at peak exercise and an increased percentage change in QT dispersion with exercise may help explain the increased susceptibility of the IHD group for arrhythmias. The cardioprotective action of beta-blockers may be explained by their blunting effect on exercise-related changes in QT dispersion.


Question: Cellular clocks are associated with mitosis timing in NIH 3T3 cells.

Phase locking and multiple oscillating attractors for the coupled mammalian clock and cell cycle.. Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

The Effect of Collinearity on Parameter Estimates in Nonlinear Mixed Effect Models. Purpose. To demonstrate how correlations among predictor variables in a population pharmacokinetic model affect the ability to discern which covariates should enter into the structural pharmacokinetic model. Methods. Monte Carlo simulation was used to generate multiple-dose concentration-time data similar to that seen in a Phase III clinical trial. The drugs' pharmacokinetics were dependent on two covariates. Five data sets were simulated with increasing correlation between the two covariates. All data sets were analyzed using NONMEM both with and without inclusion of the covariates in the structural pharmacokinetic model. Summary measures for ill-conditioning and sensitivity analysis were used to examine how increasing correlation among covariates affects the accuracy and precision of the parameter estimates. Results. When covariates were included in the structural pharmacokinetic model and the correlation between covariates increased, the standard error of the parameter estimates increased and the value of parameter estimates themselves became increasingly biased. When the correlation between predictor variables was 0.75, the standard errors of the parameter estimates were too large to declare statistical significance. Conclusions. Correlations among predictor variables greater than 0.5 when entered into the model simultaneously should be a warning to researchers because the (1) the accuracy of the parameter estimates themselves may be biased and (2) the precision of the estimates may be inflated due to ill-conditioning.


Question: Physical activity level is associated with the difference in maximal oxygen consumption between black and white youth.

Determinants of adolescent physical activity and inactivity patterns.. OBJECTIVES Despite recognition of the important influence of environmental determinants on physical activity patterns, minimal empirical research has been done to assess the impact of environmental/contextual determinants of physical activity. This article aims to investigate environmental and sociodemographic determinants of physical activity and inactivity patterns among subpopulations of US adolescents. We define environmental determinants as modifiable factors in the physical environment that impose a direct influence on the opportunity to engage in physical activity. The present research examines environmental and sociodemographic determinants of physical activity and inactivity with the implication that these findings can point toward societal-level intervention strategies for increasing physical activity and decreasing inactivity among adolescents. STUDY DESIGN AND METHODOLOGY The study population consists of nationally representative data from the 1996 National Longitudinal Study of Adolescent Health on 17 766 US adolescents enrolled in US middle and high schools (including 3933 non-Hispanic blacks, 3148 Hispanics, and 1337 Asians). Hours/week of inactivity (TV/video viewing and video/computer games) and times/week of moderate to vigorous physical activity were collected by questionnaire. Outcome variables were moderate to vigorous physical activity and inactivity, which were broken into categories (physical activity: 0-2 times/week, 3-4 times/week, and >/=5 times/week; inactivity: 0-10 hours/week, 11-24 hours/week, and >/=25 hours/week). Sociodemographic and environmental correlates of physical activity and inactivity were used as exposure and control variables and included sex, age, urban residence, participation in school physical education program, use of community recreation center, total reported incidents of serious crime in neighborhood, socioeconomic status, ethnicity, generation of residence in the United States, presence of mother/father in household, pregnancy status, work status, in-school status, region, and month of interview. Logistic regression models of high versus low and medium physical activity and inactivity were used to investigate sex and ethnic interactions in relation to environmental and sociodemographic factors to examine evidence for the potential impact of physical education and recreation programs and sociodemographic factors on physical activity and inactivity patterns. RESULTS Moderate to vigorous physical activity was lower and inactivity higher for non-Hispanic black and Hispanic adolescents. Participation in school physical education programs was considerably low for these adolescents and decreased with age. Participation in daily school physical education (PE) program classes (adjusted odds ratio [AOR]: 2.21; confidence interval [CI]: 1.82-2.68) and use of a community recreation center (AOR: 1.75; CI: 1.56-1.96) were associated with an increased likelihood of engaging in high level moderate to vigorous physical activity. Maternal education was inversely associated with high inactivity patterns; for example, having a mother with a graduate or professional degree was associated with an AOR of.61 (CI:.48-.76) for high inactivity. High family income was associated with increased moderate to vigorous physical activity (AOR: 1.43; CI: 1.22-1.67) and decreased inactivity (AOR:.70; CI:.59-.82). High neighborhood serious crime level was associated with a decreased likelihood of falling in the highest category of moderate to vigorous physical activity (AOR:.77; CI:.66-.91). CONCLUSIONS These results show important associations between modifiable environmental factors, such as participation in school PE and community recreation programs, with activity patterns of adolescents. Despite the marked and significant impact of participation in school PE programs on physical activity patterns of US adolescents, few adolescents participated in such school PE programs; only 21.3% of all adolescents

Transmission of sporadic Creutzfeldt-Jakob disease by blood transfusion: risk factor or possible biases.. BACKGROUND The occurrence of transfusion transmissions of variant Creutzfeldt-Jakob disease (CJD) cases has reawakened attention to the possible similar risk posed by other forms of CJD. STUDY DESIGN AND METHODS CJD with a definite or probable diagnosis (sporadic CJD, n = 741; genetic CJD, n = 175) and no-CJD patients with definite alternative diagnosis (n = 482) with available blood transfusion history were included in the study. The risk of exposure to blood transfusion occurring more than 10 years before disease onset and for some possible confounding factors was evaluated by calculating crude odds ratios (ORs). Variables with significant ORs in univariate analyses were included in multivariate logistic regression analyses. RESULTS In the univariate model, blood transfusion occurring more than 10 years before clinical onset is 4.1-fold more frequent in sporadic CJD than in other neurologic disorders. This significance is lost when the 10-year lag time was not considered. Multivariate analyses show that the risk of developing sporadic CJD after transfusion increases (OR, 5.05) after adjusting for possible confounding factors. Analysis conducted on patients with genetic CJD did not reveal any significant risk factor associated with transfusion. CONCLUSION This is the first case-control study showing a significant risk of transfusion occurring more than 10 years before clinical onset in sporadic CJD patients. It remains questionable whether the significance of these data is biologically plausible or the consequence of biases in the design of the study, but they counterbalance previous epidemiologic negative reports that might have overestimated the assessment of blood safety in sporadic CJD.


Question: Removal of H3K9me3 by ectopic expression of other H3K9 demethylases improves reprogramming efficiency in SCNT experiments.

H3K9 demethylase KDM4E is an epigenetic regulator for bovine embryonic development and a defective factor for nuclear reprogramming.. Aberrant epigenetic reprogramming often results in developmental defects in somatic cell nuclear transfer (SCNT) embryos during embryonic genome activation (EGA). Bovine eight-cell SCNT embryos exhibit global hypermethylation of histone H3 lysine 9 tri- and di-methylation (H3K9me3/2), but the intrinsic reason for this remains elusive. Here, we provide evidence that two H3K9 demethylase genes, lysine-specific demethylase 4D (KDM4D) and 4E (KDM4E), are related to active H3K9me3/2 demethylation in in vitro fertilized (IVF) embryos and are deficiently expressed in cloned embryos at the time of EGA. Moreover, KDM4E plays a more crucial role in IVF and SCNT embryonic development, and overexpression of KDM4E can restore the global transcriptome, improve blastocyst formation and increase the cloning efficiency of SCNT embryos. Our results thereby indicate that KDM4E can function as a crucial epigenetic regulator of EGA and as an internal defective factor responsible for persistent H3K9me3/2 barriers to SCNT-mediated reprogramming. Furthermore, we show that interactions between RNA and KDM4E are essential for H3K9 demethylation during EGA. These observations advance the understanding of incomplete nuclear reprogramming and are of great importance for transgenic cattle procreation.

Acute kidney injury and chronic kidney disease: an integrated clinical syndrome.. The previous conventional wisdom that survivors of acute kidney injury (AKI) tend to do well and fully recover renal function appears to be flawed. AKI can cause end-stage renal disease (ESRD) directly, and increase the risk of developing incident chronic kidney disease (CKD) and worsening of underlying CKD. In addition, severity, duration, and frequency of AKI appear to be important predictors of poor patient outcomes. CKD is an important risk factor for the development and ascertainment of AKI. Experimental data support the clinical observations and the bidirectional nature of the relationships between AKI and CKD. Reductions in renal mass and nephron number, vascular insufficiency, cell cycle disruption, and maladaptive repair mechanisms appear to be important modulators of progression in patients with and without coexistent CKD. Distinction between AKI and CKD may be artificial. Consideration should be given to the integrated clinical syndrome of diminished GFR, with acute and chronic stages, where spectrum of disease state and outcome is determined by host factors, including the balance of adaptive and maladaptive repair mechanisms over time. Physicians must provide long-term follow-up to patients with first episodes of AKI, even if they presented with normal renal function.


Question: Normal granulomas form in the presence of TNF in Zebrafish.

Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death.. Tumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.

HMG-CoA reductase inhibitors and the risk of hip fractures in elderly patients.. CONTEXT Recent animal studies have found that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lipid-lowering drugs (statins) substantially increase bone formation, but whether statin use in humans results in clinically meaningful bone formation or a reduction in the risk of osteoporotic fractures is not known. OBJECTIVE To determine whether the use of statins is associated with reduced hip fracture risk. DESIGN Case-control study. SETTING AND PATIENTS A total of 6110 New Jersey residents aged 65 years or older and enrolled in Medicare and either Medicaid or the Pharmacy Assistance for the Aged and Disabled program. Case patients (n=1222) underwent surgical repair of a hip fracture in 1994. Control patients (n=4888) were identified at a ratio of 4:1 and frequency-matched to case patients for age and sex. MAIN OUTCOME MEASURE Adjusted odds ratio (OR) of hip fracture by statin use in the 180 days and 3 years prior to the index date (the earliest date of admission for surgery), adjusted for demographic and clinical characteristics and health care utilization. RESULTS Use of statins in either the prior 180 days (adjusted OR, 0.50; 95% confidence interval [CI], 0.33-0.76) or prior 3 years (adjusted OR, 0.57; 95% CI, 0.40-0.82) was associated with a significant reduction in the risk of hip fracture, even after controlling for variables such as race, insurance status, psychoactive medications, estrogen and thiazide use, ischemic heart disease, cancer, and diabetes mellitus. No significant relationship was observed between use of nonstatin lipid-lowering agents and hip fracture risk. Clear relationships were observed between the degree of reduction in hip fracture risk and the extent of statin use; there was no evidence of such relationships with nonstatin lipid-lowering agents. After adjusting for extent of statin use in the prior 3 years, current use (on the index date) was associated with a 71% reduction in risk (adjusted OR, 0.29; 95% CI, 0.10-0.81). The relationship between statin use and hip fracture risk persisted after controlling for variables such as the number of medications, the Charlson comorbidity index score, and hospitalization or nursing home stay in the last 180 days, as well as after excluding patients who were in a nursing home prior to their index date or who died in the year after their index date. Use of nonstatin lipid-lowering agents was not observed to be associated with reduction in hip fracture risk in any of these alternative models or analyses. CONCLUSIONS These findings support an association between statin use by elderly patients and reduction in the risk of hip fracture. Controlled trials are needed to exclude the possibility of unmeasured confounders. JAMA. 2000;283:3211-3216


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

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

Rapid helper T-cell recovery above 200 × 106/l at 3 months correlates to successful transplant outcomes after allogeneic stem cell transplantation. The current study evaluates the role of quantitative measurement of peripheral lymphocyte subsets, especially CD4+ helper T-cell recovery, in predicting transplant outcomes including overall survival (OS) and non-relapse mortality (NRM) after allogeneic stem cell transplantation. A total of 69 allogeneic recipients were included with following diagnoses: acute myeloid leukemia 42, acute lymphoblastic leukemia 5, chronic myeloid leukemia 15, non-Hodgkin's lymphoma 5 and high-risk myelodysplastic syndrome 2. The peripheral lymphocyte subset counts (CD3+ T cells, CD3+4+ helper T cells, CD3+8+ cytotoxic T cells, CD19+ B cells, and CD56+ natural killer cells) were measured at 3, 6 and 12 months. The CD4+ helper T-cell reconstitution at 3 months was strongly correlated with OS (P<0.0001), NRM (P=0.0007), and opportunistic infections (P=0.0108) at the cutoff value of 200 × 106/l CD4+ helper T cells. Rapid CD4+ helper T-cell recovery was also associated with a higher CD4+ helper T-cell transplant dose (P=0.006) and donor type (P<0.001). An early CD4+ helper T-cell recovery at 3 months correlated with a subsequent faster helper T-cell recovery until 12 months, yet not with B-cell recovery. In a multivariate analysis, rapid recovery of CD4+ helper T cells at 3 months was a favorable prognostic factor together with higher CD34+ cell transplant dose in terms of OS (P=0.001) and NRM (P=0.005).


Question: Pro-inflammatory cytokines are up regulated during tumor development.

Paraneoplastic thrombocytosis: the secrets of tumor self-promotion.. Paraneoplastic thrombocytosis is associated with many solid tumors and often correlates with reduced survival. Recent studies suggest that a pathogenic feed back loop may be operative between platelets and tumor cells, with reciprocal interactions between tumor growth/metastasis and thrombocytosis/platelet activation. Specific molecular pathways have been identified in which tumors can stimulate platelet production and activation; activated platelets can, in turn, promote tumor growth and metastasis. Taken together, these findings provide exciting new potential targets for therapeutic intervention.

Loss, trauma, and human resilience: have we underestimated the human capacity to thrive after extremely aversive events?. Many people are exposed to loss or potentially traumatic events at some point in their lives, and yet they continue to have positive emotional experiences and show only minor and transient disruptions in their ability to function. Unfortunately, because much of psychology's knowledge about how adults cope with loss or trauma has come from individuals who sought treatment or exhibited great distress, loss and trauma theorists have often viewed this type of resilience as either rare or pathological. The author challenges these assumptions by reviewing evidence that resilience represents a distinct trajectory from the process of recovery, that resilience in the face of loss or potential trauma is more common than is often believed, and that there are multiple and sometimes unexpected pathways to resilience.


Question: Beta-band coherence is enhanced for visible stimuli over invisible stimuli.

Converging Intracranial Markers of Conscious Access. We compared conscious and nonconscious processing of briefly flashed words using a visual masking procedure while recording intracranial electroencephalogram (iEEG) in ten patients. Nonconscious processing of masked words was observed in multiple cortical areas, mostly within an early time window (<300 ms), accompanied by induced gamma-band activity, but without coherent long-distance neural activity, suggesting a quickly dissipating feedforward wave. In contrast, conscious processing of unmasked words was characterized by the convergence of four distinct neurophysiological markers: sustained voltage changes, particularly in prefrontal cortex, large increases in spectral power in the gamma band, increases in long-distance phase synchrony in the beta range, and increases in long-range Granger causality. We argue that all of those measures provide distinct windows into the same distributed state of conscious processing. These results have a direct impact on current theoretical discussions concerning the neural correlates of conscious access.

Transnationalism and identity. Transnationalism and identity are concepts that inherently call for juxtaposition. This is so because many peoples' transnational networks of exchange and participation are grounded upon some perception of common identity; conversely, the identities of numerous individuals and groups of people are negotiated within social worlds that span more than one place. In this introductory article, the transnational perspective on migration studies is first discussed, followed by some critiques and outstanding questions. The final section summarises points raised by the contributing authors of the main articles in this themed issue of JEMS, especially with regard to various ways transnational settings and dynamics affect the construction, negotiation and reproduction of identities.


Question: Roughly 55% of women with chronic pelvic pain have no underlying pathology.

Laparoscopic uterosacral nerve ablation for alleviating chronic pelvic pain: a randomized controlled trial.. CONTEXT Chronic pelvic pain is a common condition with a major effect on health-related quality of life, work productivity, and health care use. Operative interruption of nerve trunks in the uterosacral ligaments by laparoscopic uterosacral nerve ablation (LUNA) is a treatment option for patients with chronic pelvic pain. OBJECTIVE To assess the effectiveness of LUNA in patients with chronic pelvic pain. DESIGN, SETTING, AND PARTICIPANTS Randomized controlled trial of 487 women with chronic pelvic pain lasting longer than 6 months without or with minimal endometriosis, adhesions, or pelvic inflammatory disease, who were recruited to the study by consultant gynecological surgeons from 18 UK hospitals between February 1998 and December 2005. Follow-up was conducted by questionnaires mailed at 3 and 6 months and at 1, 2, 3, and 5 years. INTERVENTION Bilateral LUNA or laparoscopy without pelvic denervation (no LUNA); participants were blinded to the treatment allocation. MAIN OUTCOME MEASURES The primary outcome was pain, which was assessed by a visual analogue scale. Data concerning the 3 types of pain (noncyclical pain, dysmenorrhea, and dyspareunia) were analyzed separately as was the worst pain level experienced from any of these 3 types of pain. The secondary outcome was health-related quality of life, which was measured using a generic instrument (EuroQoL EQ-5D and EQ-VAS). RESULTS After a median follow-up of 69 months, there were no significant differences reported on the visual analogue pain scales for the worst pain (mean difference between the LUNA group and the no LUNA group, -0.04 cm [95% confidence interval {CI}, -0.33 to 0.25 cm]; P = .80), noncyclical pain (-0.11 cm [95% CI, -0.50 to 0.29 cm]; P = .60), dysmenorrhea (-0.09 cm [95% CI, -0.49 to 0.30 cm]; P = .60), or dyspareunia (0.18 cm [95% CI, -0.22 to 0.62 cm]; P = .40). No differences were observed between the LUNA group and the no LUNA group for quality of life. CONCLUSION Among women with chronic pelvic pain, LUNA did not result in improvements in pain, dysmenorrhea, dyspareunia, or quality of life compared with laparoscopy without pelvic denervation. TRIAL REGISTRATION controlled-trials.com Identifier: ISRCTN41196151.

Lipoxin a4 analogs attenuate induction of intestinal epithelial proinflammatory gene expression and reduce the severity of dextran sodium sulfate-induced colitis.. The anti-inflammatory eicosanoid lipoxin A(4) (LXA(4)), aspirin-triggered 15-epi-LXA(4), and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA(4) analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA(4) analog pretreatment attenuated induction of approximately 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA(4) analog pretreatment is regulated by NF-kappaB, suggesting that LXA(4) analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA(4) analog reduced NF-kappaB-mediated transcriptional activation in a LXA(4) receptor-dependent manner and inhibited induced degradation of IkappaBalpha. LXA(4) analog did not affect earlier stimulus-induced signaling events that lead to IkappaBalpha degradation, such as S. typhimurium-induced epithelial Ca(2+) mobilization or TNF-alpha-induced phosphorylation of IkappaBalpha. To establish the in vivo relevance of these findings, we examined whether LXA(4) analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA(4) analog (15-epi-16-para-fluoro-phenoxy-LXA(4), 10 microg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA(4) analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-kappaB pathway can be therapeutic for diseases characterized by mucosal inflammation.


Question: Environmentally-induced senescence is mimicked in disease modeling by adding specific molecules to the culture system.

Human iPSC-based modeling of late-onset disease via progerin-induced aging.. Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.

Vascular Calcifying Progenitor Cells Possess Bidirectional Differentiation Potentials. Vascular calcification is an advanced feature of atherosclerosis for which no effective therapy is available. To investigate the modulation or reversal of calcification, we identified calcifying progenitor cells and investigated their calcifying/decalcifying potentials. Cells from the aortas of mice were sorted into four groups using Sca-1 and PDGFRα markers. Sca-1(+) (Sca-1(+)/PDGFRα(+) and Sca-1(+)/PDGFRα(-)) progenitor cells exhibited greater osteoblastic differentiation potentials than Sca-1(-) (Sca-1(-)/PDGFRα(+) and Sca-1(-)/PDGFRα(-)) progenitor cells. Among Sca-1(+) progenitor populations, Sca-1(+)/PDGFRα(-) cells possessed bidirectional differentiation potentials towards both osteoblastic and osteoclastic lineages, whereas Sca-1(+)/PDGFRα(+) cells differentiated into an osteoblastic lineage unidirectionally. When treated with a peroxisome proliferator activated receptor γ (PPARγ) agonist, Sca-1(+)/PDGFRα(-) cells preferentially differentiated into osteoclast-like cells. Sca-1(+) progenitor cells in the artery originated from the bone marrow (BM) and could be clonally expanded. Vessel-resident BM-derived Sca-1(+) calcifying progenitor cells displayed nonhematopoietic, mesenchymal characteristics. To evaluate the modulation of in vivo calcification, we established models of ectopic and atherosclerotic calcification. Computed tomography indicated that Sca-1(+) progenitor cells increased the volume and calcium scores of ectopic calcification. However, Sca-1(+)/PDGFRα(-) cells treated with a PPARγ agonist decreased bone formation 2-fold compared with untreated cells. Systemic infusion of Sca-1(+)/PDGFRα(-) cells into Apoe(-/-) mice increased the severity of calcified atherosclerotic plaques. However, Sca-1(+)/PDGFRα(-) cells in which PPARγ was activated displayed markedly decreased plaque severity. Immunofluorescent staining indicated that Sca-1(+)/PDGFRα(-) cells mainly expressed osteocalcin; however, activation of PPARγ triggered receptor activator for nuclear factor-κB (RANK) expression, indicating their bidirectional fate in vivo. These findings suggest that a subtype of BM-derived and vessel-resident progenitor cells offer a therapeutic target for the prevention of vascular calcification and that PPARγ activation may be an option to reverse calcification.


Question: Increased microtubule acetylation worsens interference of axonal transport caused by LRRK2 Roc-COR domain mutations.

Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations. Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson's disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson's disease.

Frontrunners of T cell activation: Initial, localized Ca2+ signals mediated by NAADP and the type 1 ryanodine receptor. The activation of T cells is the fundamental on switch for the adaptive immune system. Ca2+ signaling is essential for T cell activation and starts as initial, short-lived, localized Ca2+ signals. The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) forms rapidly upon T cell activation and stimulates early Ca2+ signaling. We developed a high-resolution imaging technique using multiple fluorescent Ca2+ indicator dyes to characterize these early signaling events and investigate the channels involved in NAADP-dependent Ca2+ signals. In the first seconds of activation of either primary murine T cells or human Jurkat cells with beads coated with an antibody against CD3, we detected Ca2+ signals with diameters close to the limit of detection and that were close to the activation site at the plasma membrane. In Jurkat cells in which the ryanodine receptor (RyR) was knocked down or in primary T cells from RyR1−/− mice, either these early Ca2+ signals were not detected or the number of signals was markedly reduced. Local Ca2+ signals observed within 20 ms upon microinjection of Jurkat cells with NAADP were also sensitive to RyR knockdown. In contrast, TRPM2 (transient receptor potential channel, subtype melastatin 2), a potential NAADP target channel, was not required for the formation of initial Ca2+ signals in primary T cells. Thus, through our high-resolution imaging method, we characterized early Ca2+ release events in T cells and obtained evidence for the involvement of RyR and NAADP in such signals.