In young and middle-aged adults, current or remote uses of ADHD medications increase the risk of serious cardiovascular events.

Experience gained from the Global Malaria Eradication Program (1955-72) identified a set of shared technical and operational factors that enabled some countries to successfully eliminate malaria. Spatial data for these factors were assembled for all malaria-endemic countries and combined to provide an objective, relative ranking of countries by technical, operational, and combined elimination feasibility. The analysis was done separately for Plasmodium falciparum and Plasmodium vivax, and the limitations of the approach were discussed. The relative rankings suggested that malaria elimination would be most feasible in countries in the Americas and Asia, and least feasible in countries in central and west Africa. The results differed when feasibility was measured by technical or operational factors, highlighting the different types of challenge faced by each country. The results are not intended to be prescriptive, predictive, or to provide absolute assessments of feasibility, but they do show that spatial information is available to facilitate evidence-based assessments of the relative feasibility of malaria elimination by country that can be rapidly updated.

The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs could possess a regulatory role that relies on their ability to compete for microRNA binding, independently of their protein-coding function. As a model for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene PTENP1 and the critical consequences of this interaction. We find that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role. We also show that the PTENP1 locus is selectively lost in human cancer. We extended our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. We also demonstrate that the transcripts of protein-coding genes such as PTEN are biologically active. These findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs.

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized by lymphocytic infiltration into nonlymphoid tissues. Here, we demonstrate that the CD28 co-stimulatory pathway regulates the trafficking of self-reactive Ctla4(-/-) T cells to tissues. Concurrent ablation of the CD28-activated Tec family kinase ITK does not block spontaneous T cell activation but instead causes self-reactive Ctla4(-/-) T cells to accumulate in secondary lymphoid organs. Despite excessive spontaneous T cell activation and proliferation in lymphoid organs, Itk(-/-); Ctla4(-/-) mice are otherwise healthy, mount antiviral immune responses and exhibit a long lifespan. We propose that ITK specifically licenses autoreactive T cells to enter tissues to mount destructive immune responses. Notably, ITK inhibitors mimic the null mutant phenotype and also prevent pancreatic islet infiltration by diabetogenic T cells in mouse models of type 1 diabetes, highlighting their potential utility for the treatment of human autoimmune disorders.

The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2–mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

BACKGROUND The diagnosis of hypertension has traditionally been based on blood-pressure measurements in the clinic, but home and ambulatory measurements better correlate with cardiovascular outcome, and ambulatory monitoring is more accurate than both clinic and home monitoring in diagnosing hypertension. We aimed to compare the cost-effectiveness of different diagnostic strategies for hypertension. METHODS We did a Markov model-based probabilistic cost-effectiveness analysis. We used a hypothetical primary-care population aged 40 years or older with a screening blood-pressure measurement greater than 140/90 mm Hg and risk-factor prevalence equivalent to the general population. We compared three diagnostic strategies-further blood pressure measurement in the clinic, at home, and with an ambulatory monitor-in terms of lifetime costs, quality-adjusted life years, and cost-effectiveness. FINDINGS Ambulatory monitoring was the most cost-effective strategy for the diagnosis of hypertension for men and women of all ages. It was cost-saving for all groups (from -£56 [95% CI -105 to -10] in men aged 75 years to -£323 [-389 to -222] in women aged 40 years) and resulted in more quality-adjusted life years for men and women older than 50 years (from 0·006 [0·000 to 0·015] for women aged 60 years to 0·022 [0·012 to 0·035] for men aged 70 years). This finding was robust when assessed with a wide range of deterministic sensitivity analyses around the base case, but was sensitive if home monitoring was judged to have equal test performance to ambulatory monitoring or if treatment was judged effective irrespective of whether an individual was hypertensive. INTERPRETATION Ambulatory monitoring as a diagnostic strategy for hypertension after an initial raised reading in the clinic would reduce misdiagnosis and save costs. Additional costs from ambulatory monitoring are counterbalanced by cost savings from better targeted treatment. Ambulatory monitoring is recommended for most patients before the start of antihypertensive drugs. FUNDING National Institute for Health Research and the National Institute for Health and Clinical Excellence.

OBJECTIVE To determine whether there are risk factors in a doctor's time at medical school that are associated with subsequent professional misconduct. DESIGN Matched case-control study. Setting Records from medical schools and the General Medical Council (GMC). PARTICIPANTS 59 doctors who had graduated from any one of eight medical schools in the United Kingdom in 1958-97 and had a proved finding of serious professional misconduct in GMC proceedings in 1999-2004 (cases); 236 controls (four for each case) were selected by systematic sampling from matching graduation cohorts. Case-control status was revealed by the GMC after completion of data entry. MAIN OUTCOME MEASURE Odds ratios for being a "case," with multivariable conditional logistic regression of potential risk factors including pre-admission characteristics and progress during the course. These data were obtained from anonymised copies of the students' progress files held by their original medical schools. RESULTS Univariate conditional logistic regression analysis found that cases were more likely to be men, to be of lower estimated social class, and to have had academic difficulties during their medical course, especially in the early years. Multivariable analysis showed that male sex (odds ratio 9.80, 95% confidence interval 2.43 to 39.44, P=0.001), lower social class (4.28, 1.52 to 12.09, P=0.006), and failure of early or preclinical examinations (5.47, 2.17 to 13.79, P<0.001) were independently associated with being a case. CONCLUSIONS This small study suggests that male sex, a lower socioeconomic background, and early academic difficulties at medical school could be risk factors for subsequent professional misconduct. The findings are preliminary and should be interpreted with caution. Most doctors with risk factors will not come before the GMC's disciplinary panels.


Question:
Ten percent (10%) of kidney cancers in the UK are attributable to excess weight.

BACKGROUND High body-mass index (BMI) predisposes to several site-specific cancers, but a large-scale systematic and detailed characterisation of patterns of risk across all common cancers adjusted for potential confounders has not previously been undertaken. We aimed to investigate the links between BMI and the most common site-specific cancers. METHODS With primary care data from individuals in the Clinical Practice Research Datalink with BMI data, we fitted Cox models to investigate associations between BMI and 22 of the most common cancers, adjusting for potential confounders. We fitted linear then non-linear (spline) models; investigated effect modification by sex, menopausal status, smoking, and age; and calculated population effects. FINDINGS 5·24 million individuals were included; 166,955 developed cancers of interest. BMI was associated with 17 of 22 cancers, but effects varied substantially by site. Each 5 kg/m(2) increase in BMI was roughly linearly associated with cancers of the uterus (hazard ratio [HR] 1·62, 99% CI 1·56-1·69; p<0·0001), gallbladder (1·31, 1·12-1·52; p<0·0001), kidney (1·25, 1·17-1·33; p<0·0001), cervix (1·10, 1·03-1·17; p=0·00035), thyroid (1·09, 1·00-1·19; p=0·0088), and leukaemia (1·09, 1·05-1·13; p≤0·0001). BMI was positively associated with liver (1·19, 1·12-1·27), colon (1·10, 1·07-1·13), ovarian (1·09, 1.04-1.14), and postmenopausal breast cancers (1·05, 1·03-1·07) overall (all p<0·0001), but these effects varied by underlying BMI or individual-level characteristics. We estimated inverse associations with prostate and premenopausal breast cancer risk, both overall (prostate 0·98, 0·95-1·00; premenopausal breast cancer 0·89, 0·86-0·92) and in never-smokers (prostate 0·96, 0·93-0·99; premenopausal breast cancer 0·89, 0·85-0·94). By contrast, for lung and oral cavity cancer, we observed no association in never smokers (lung 0·99, 0·93-1·05; oral cavity 1·07, 0·91-1·26): inverse associations overall were driven by current smokers and ex-smokers, probably because of residual confounding by smoking amount. Assuming causality, 41% of uterine and 10% or more of gallbladder, kidney, liver, and colon cancers could be attributable to excess weight. We estimated that a 1 kg/m(2) population-wide increase in BMI would result in 3790 additional annual UK patients developing one of the ten cancers positively associated with BMI. INTERPRETATION BMI is associated with cancer risk, with substantial population-level effects. The heterogeneity in the effects suggests that different mechanisms are associated with different cancer sites and different patient subgroups. FUNDING National Institute for Health Research, Wellcome Trust, and Medical Research Council.

Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.

To characterize the properties of adult neural stem cells (NSCs), we generated and analyzed Sox2-GFP transgenic mice. Sox2-GFP cells in the subgranular zone (SGZ) express markers specific for progenitors, but they represent two morphologically distinct populations that differ in proliferation levels. Lentivirus- and retrovirus-mediated fate-tracing studies showed that Sox2+ cells in the SGZ have potential to give rise to neurons and astrocytes, revealing their multipotency at the population as well as at a single-cell level. A subpopulation of Sox2+ cells gives rise to cells that retain Sox2, highlighting Sox2+ cells as a primary source for adult NSCs. In response to mitotic signals, increased proliferation of Sox2+ cells is coupled with the generation of Sox2+ NSCs as well as neuronal precursors. An asymmetric contribution of Sox2+ NSCs may play an important role in maintaining the constant size of the NSC pool and producing newly born neurons during adult neurogenesis.

The overall size and structure of a synaptic terminal is an important determinant of its function. In a large-scale mutagenesis screen, designed to identify Drosophila mutants with abnormally structured neuromuscular junctions (NMJs), we discovered mutations in Drosophila mical, a conserved gene encoding a multi-domain protein with a N-terminal monooxygenase domain. In mical mutants, synaptic boutons do not sprout normally over the muscle surface and tend to form clusters along synaptic branches and at nerve entry sites. Consistent with high expression of MICAL in somatic muscles, immunohistochemical stainings reveal that the subcellular localization and architecture of contractile muscle filaments are dramatically disturbed in mical mutants. Instead of being integrated into a regular sarcomeric pattern, actin and myosin filaments are disorganized and accumulate beneath the plasmamembrane. Whereas contractile elements are strongly deranged, the proposed organizer of sarcomeric structure, D-Titin, is much less affected. Transgenic expression of interfering RNA molecules demonstrates that MICAL is required in muscles for the higher order arrangement of myofilaments. Ultrastructural analysis confirms that myosin-rich thick filaments enter submembranous regions and interfere with synaptic development, indicating that the disorganized myofilaments may cause the synaptic growth phenotype. As a model, we suggest that the filamentous network around synaptic boutons restrains the spreading of synaptic branches.


Question:
Antiretroviral therapy increases rates of tuberculosis across a broad range of CD4 strata.

In this study, we used whole-genome sequencing and gene expression profiling of 215 human induced pluripotent stem cell (iPSC) lines from different donors to identify genetic variants associated with RNA expression for 5,746 genes. We were able to predict causal variants for these expression quantitative trait loci (eQTLs) that disrupt transcription factor binding and validated a subset of them experimentally. We also identified copy-number variant (CNV) eQTLs, including some that appear to affect gene expression by altering the copy number of intergenic regulatory regions. In addition, we were able to identify effects on gene expression of rare genic CNVs and regulatory single-nucleotide variants and found that reactivation of gene expression on the X chromosome depends on gene chromosomal position. Our work highlights the value of iPSCs for genetic association analyses and provides a unique resource for investigating the genetic regulation of gene expression in pluripotent cells.

Understanding the genetic basis of adaptation is a central problem in biology. However, revealing the underlying molecular mechanisms has been challenging as changes in fitness may result from perturbations to many pathways, any of which may contribute relatively little. We have developed a combined experimental/computational framework to address this problem and used it to understand the genetic basis of ethanol tolerance in Escherichia coli. We used fitness profiling to measure the consequences of single-locus perturbations in the context of ethanol exposure. A module-level computational analysis was then used to reveal the organization of the contributing loci into cellular processes and regulatory pathways (e.g. osmoregulation and cell-wall biogenesis) whose modifications significantly affect ethanol tolerance. Strikingly, we discovered that a dominant component of adaptation involves metabolic rewiring that boosts intracellular ethanol degradation and assimilation. Through phenotypic and metabolomic analysis of laboratory-evolved ethanol-tolerant strains, we investigated naturally accessible pathways of ethanol tolerance. Remarkably, these laboratory-evolved strains, by and large, follow the same adaptive paths as inferred from our coarse-grained search of the fitness landscape.

BACKGROUND Although cigarette smoking, excessive alcohol drinking, obesity, and several other well-studied unhealthy lifestyle-related factors each have been linked to the risk of multiple chronic diseases and premature death, little is known about the combined impact on mortality outcomes, in particular among Chinese and other non-Western populations. The objective of this study was to quantify the overall impact of lifestyle-related factors beyond that of active cigarette smoking and alcohol consumption on all-cause and cause-specific mortality in Chinese women. METHODS AND FINDINGS We used data from the Shanghai Women's Health Study, an ongoing population-based prospective cohort study in China. Participants included 71,243 women aged 40 to 70 years enrolled during 1996-2000 who never smoked or drank alcohol regularly. A healthy lifestyle score was created on the basis of five lifestyle-related factors shown to be independently associated with mortality outcomes (normal weight, lower waist-hip ratio, daily exercise, never exposed to spouse's smoking, higher daily fruit and vegetable intake). The score ranged from zero (least healthy) to five (most healthy) points. During an average follow-up of 9 years, 2,860 deaths occurred, including 775 from cardiovascular disease (CVD) and 1,351 from cancer. Adjusted hazard ratios for mortality decreased progressively with an increasing number of healthy lifestyle factors. Compared to women with a score of zero, hazard ratios (95% confidence intervals) for women with four to five factors were 0.57 (0.44-0.74) for total mortality, 0.29 (0.16-0.54) for CVD mortality, and 0.76 (0.54-1.06) for cancer mortality. The inverse association between the healthy lifestyle score and mortality was seen consistently regardless of chronic disease status at baseline. The population attributable risks for not having 4-5 healthy lifestyle factors were 33% for total deaths, 59% for CVD deaths, and 19% for cancer deaths. CONCLUSIONS In this first study, to our knowledge, to quantify the combined impact of lifestyle-related factors on mortality outcomes in Chinese women, a healthier lifestyle pattern-including being of normal weight, lower central adiposity, participation in physical activity, nonexposure to spousal smoking, and higher fruit and vegetable intake-was associated with reductions in total and cause-specific mortality among lifetime nonsmoking and nondrinking women, supporting the importance of overall lifestyle modification in disease prevention. Please see later in the article for the Editors' Summary.

Hepatic stellate cells (HSCs) play critical roles in liver fibrosis and hepatocellular carcinoma (HCC). Vitamin D receptor (VDR) activation in HSCs inhibits liver inflammation and fibrosis. We found that p62/SQSTM1, a protein upregulated in liver parenchymal cells but downregulated in HCC-associated HSCs, negatively controls HSC activation. Total body or HSC-specific p62 ablation potentiates HSCs and enhances inflammation, fibrosis, and HCC progression. p62 directly interacts with VDR and RXR promoting their heterodimerization, which is critical for VDR:RXR target gene recruitment. Loss of p62 in HSCs impairs the repression of fibrosis and inflammation by VDR agonists. This demonstrates that p62 is a negative regulator of liver inflammation and fibrosis through its ability to promote VDR signaling in HSCs, whose activation supports HCC.

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

Fibrosis is a pathological result of a dysfunctional repair response to tissue injury and occurs in a number of organs, including the lungs1. Cellular metabolism regulates tissue repair and remodelling responses to injury2-4. AMPK is a critical sensor of cellular bioenergetics and controls the switch from anabolic to catabolic metabolism5. However, the role of AMPK in fibrosis is not well understood. Here, we demonstrate that in humans with idiopathic pulmonary fibrosis (IPF) and in an experimental mouse model of lung fibrosis, AMPK activity is lower in fibrotic regions associated with metabolically active and apoptosis-resistant myofibroblasts. Pharmacological activation of AMPK in myofibroblasts from lungs of humans with IPF display lower fibrotic activity, along with enhanced mitochondrial biogenesis and normalization of sensitivity to apoptosis. In a bleomycin model of lung fibrosis in mice, metformin therapeutically accelerates the resolution of well-established fibrosis in an AMPK-dependent manner. These studies implicate deficient AMPK activation in non-resolving, pathologic fibrotic processes, and support a role for metformin (or other AMPK activators) to reverse established fibrosis by facilitating deactivation and apoptosis of myofibroblasts.


Rising temperatures caused by global warming increases risk of dengue fever transmission.

BACKGROUND Transmission of dengue viruses (DENV), the leading cause of arboviral disease worldwide, is known to vary through time and space, likely owing to a combination of factors related to the human host, virus, mosquito vector, and environment. An improved understanding of variation in transmission patterns is fundamental to conducting surveillance and implementing disease prevention strategies. To test the hypothesis that DENV transmission is spatially and temporally focal, we compared geographic and temporal characteristics within Thai villages where DENV are and are not being actively transmitted. METHODS AND FINDINGS Cluster investigations were conducted within 100 m of homes where febrile index children with (positive clusters) and without (negative clusters) acute dengue lived during two seasons of peak DENV transmission. Data on human infection and mosquito infection/density were examined to precisely (1) define the spatial and temporal dimensions of DENV transmission, (2) correlate these factors with variation in DENV transmission, and (3) determine the burden of inapparent and symptomatic infections. Among 556 village children enrolled as neighbors of 12 dengue-positive and 22 dengue-negative index cases, all 27 DENV infections (4.9% of enrollees) occurred in positive clusters (p < 0.01; attributable risk [AR] = 10.4 per 100; 95% confidence interval 1-19.8 per 100]. In positive clusters, 12.4% of enrollees became infected in a 15-d period and DENV infections were aggregated centrally near homes of index cases. As only 1 of 217 pairs of serologic specimens tested in positive clusters revealed a recent DENV infection that occurred prior to cluster initiation, we attribute the observed DENV transmission subsequent to cluster investigation to recent DENV transmission activity. Of the 1,022 female adult Ae. aegypti collected, all eight (0.8%) dengue-infected mosquitoes came from houses in positive clusters; none from control clusters or schools. Distinguishing features between positive and negative clusters were greater availability of piped water in negative clusters (p < 0.01) and greater number of Ae. aegypti pupae per person in positive clusters (p = 0.04). During primarily DENV-4 transmission seasons, the ratio of inapparent to symptomatic infections was nearly 1:1 among child enrollees. Study limitations included inability to sample all children and mosquitoes within each cluster and our reliance on serologic rather than virologic evidence of interval infections in enrollees given restrictions on the frequency of blood collections in children. CONCLUSIONS Our data reveal the remarkably focal nature of DENV transmission within a hyperendemic rural area of Thailand. These data suggest that active school-based dengue case detection prompting local spraying could contain recent virus introductions and reduce the longitudinal risk of virus spread within rural areas. Our results should prompt future cluster studies to explore how host immune and behavioral aspects may impact DENV transmission and prevention strategies. Cluster methodology could serve as a useful research tool for investigation of other temporally and spatially clustered infectious diseases.

BACKGROUND Information is scarce about the combined effects on breast cancer incidence of low-penetrance genetic susceptibility polymorphisms and environmental factors (reproductive, behavioural, and anthropometric risk factors for breast cancer). To test for evidence of gene-environment interactions, we compared genotypic relative risks for breast cancer across the other risk factors in a large UK prospective study. METHODS We tested gene-environment interactions in 7610 women who developed breast cancer and 10 196 controls without the disease, studying the effects of 12 polymorphisms (FGFR2-rs2981582, TNRC9-rs3803662, 2q35-rs13387042, MAP3K1-rs889312, 8q24-rs13281615, 2p-rs4666451, 5p12-rs981782, CASP8-rs1045485, LSP1-rs3817198, 5q-rs30099, TGFB1-rs1982073, and ATM-rs1800054) in relation to prospectively collected information about ten established environmental risk factors (age at menarche, parity, age at first birth, breastfeeding, menopausal status, age at menopause, use of hormone replacement therapy, body-mass index, height, and alcohol consumption). FINDINGS After allowance for multiple testing none of the 120 comparisons yielded significant evidence of a gene-environment interaction. By contrast with previous suggestions, there was little evidence that the genotypic relative risks were affected by use of hormone replacement therapy, either overall or for oestrogen-receptor-positive disease. Only one of the 12 polymorphisms was correlated with any of the ten other risk factors: carriers of the high-risk C allele of MAP3K1-rs889312 were significantly shorter than non-carriers (mean height 162.4 cm [95% CI 162.1-162.7] vs 163.1 cm [162.9-163.2]; p=0.01 after allowance for multiple testing). INTERPRETATION Risks of breast cancer associated with low-penetrance susceptibility polymorphisms do not vary significantly with these ten established environmental risk factors. FUNDING Cancer Research UK and the UK Medical Research Council.

Mitochondria are the primary energy-generating system in most eukaryotic cells. Additionally, they participate in intermediary metabolism, calcium signaling, and apoptosis. Given these well-established functions, it might be expected that mitochondrial dysfunction would give rise to a simple and predictable set of defects in all tissues. However, mitochondrial dysfunction has pleiotropic effects in multicellular organisms. Clearly, much about the basic biology of mitochondria remains to be understood. Here we discuss recent work that suggests that the dynamics (fusion and fission) of these organelles is important in development and disease.

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.

Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

CONTEXT Adequate vitamin D status for optimum bone health has received increased recognition in recent years; however, the ideal intake is not known. Serum 25-hydroxyvitamin D is the generally accepted indicator of vitamin D status, but no universal reference level has been reached. OBJECTIVE To investigate the relative importance of high calcium intake and serum 25-hydroxyvitamin D for calcium homeostasis, as determined by serum intact parathyroid hormone (PTH). DESIGN, SETTING, AND PARTICIPANTS Cross-sectional study of 2310 healthy Icelandic adults who were divided equally into 3 age groups (30-45 years, 50-65 years, or 70-85 years) and recruited from February 2001 to January 2003. They were administered a semi-quantitative food frequency questionnaire, which assessed vitamin D and calcium intake. Participants were further divided into groups according to calcium intake (<800 mg/d, 800-1200 mg/d, and >1200 mg/d) and serum 25-hydroxyvitamin D level (<10 ng/mL, 10-18 ng/mL, and >18 ng/mL). MAIN OUTCOME MEASURE Serum intact PTH as determined by calcium intake and vitamin D.   RESULTS A total of 944 healthy participants completed all parts of the study. After adjusting for relevant factors, serum PTH was lowest in the group with a serum 25-hydroxyvitamin D level of more than 18 ng/mL but highest in the group with a serum 25-hydroxyvitamin D level of less than 10 ng/mL. At the low serum 25-hydroxyvitamin D level (<10 ng/mL), calcium intake of less than 800 mg/d vs more than 1200 mg/d was significantly associated with higher serum PTH (P = .04); and at a calcium intake of more than 1200 mg/d, there was a significant difference between the lowest and highest vitamin D groups (P = .04). CONCLUSIONS As long as vitamin D status is ensured, calcium intake levels of more than 800 mg/d may be unnecessary for maintaining calcium metabolism. Vitamin D supplements are necessary for adequate vitamin D status in northern climates.


Question:
Splenomegaly is observed in knockin mouse lacking the SHP-2 MAPK pathway.

Podocyte dysfunction, represented by foot process effacement and proteinuria, is often the starting point for progressive kidney disease. Therapies aimed at the cellular level of the disease are currently not available. Here we show that induction of urokinase receptor (uPAR) signaling in podocytes leads to foot process effacement and urinary protein loss via a mechanism that includes lipid-dependent activation of αvβ3 integrin. Mice lacking uPAR (Plaur−/−) are protected from lipopolysaccharide (LPS)-mediated proteinuria but develop disease after expression of a constitutively active β3 integrin. Gene transfer studies reveal a prerequisite for uPAR expression in podocytes, but not in endothelial cells, for the development of LPS-mediated proteinuria. Mechanistically, uPAR is required to activate αvβ3 integrin in podocytes, promoting cell motility and activation of the small GTPases Cdc42 and Rac1. Blockade of αvβ3 integrin reduces podocyte motility in vitro and lowers proteinuria in mice. Our findings show a physiological role for uPAR signaling in the regulation of kidney permeability.

Aire-expressing medullary thymic epithelial cells (mTECs) play a key role in preventing autoimmunity by expressing tissue-restricted antigens to help purge the emerging T cell receptor repertoire of self-reactive specificities. Here we demonstrate a novel role for a CD4+3− inducer cell population, previously linked to development of organized secondary lymphoid structures and maintenance of T cell memory in the functional regulation of Aire-mediated promiscuous gene expression in the thymus. CD4+3− cells are closely associated with mTECs in adult thymus, and in fetal thymus their appearance is temporally linked with the appearance of Aire+ mTECs. We show that RANKL signals from this cell promote the maturation of RANK-expressing CD80−Aire− mTEC progenitors into CD80+Aire+ mTECs, and that transplantation of RANK-deficient thymic stroma into immunodeficient hosts induces autoimmunity. Collectively, our data reveal cellular and molecular mechanisms leading to the generation of Aire+ mTECs and highlight a previously unrecognized role for CD4+3−RANKL+ inducer cells in intrathymic self-tolerance.

We generated a series of knockin mouse lines, in which the cytokine receptor gp130-dependent STAT3 and/or SHP2 signals were disrupted, by replacing the mouse gp130 gene with human gp130 mutant cDNAs. The SHP2 signal-deficient mice (gp130F759/F759 were born normal but displayed splenomegaly and lymphadenopathy and an enhanced acute phase reaction. In contrast, the STAT3 signal-deficient mice (gp130FXQ/FXXQ) died perinatally, like the gp130-deficient mice (gp130D/D). The gp130F759/F759 mice showed prolonged gp130-induced STAT3 activation, indicating a negative regulatory role for SHP2. Th1-type cytokine production and IgG2a and IgG2b production were increased in the gp130F759/F759 mice, while they were decreased in the gp130FXXQ/FXXQ immune system. These results indicate that the balance of positive and negative signals generated through gp130 regulates the immune responses.

Self-renewal and differentiation of stem cells depend on asymmetric division and polarized motility processes that in other cell types are modulated by nonmuscle myosin-II (MII) forces and matrix mechanics. Here, mass spectrometry-calibrated intracellular flow cytometry of human hematopoiesis reveals MIIB to be a major isoform that is strongly polarized in hematopoietic stem cells and progenitors (HSC/Ps) and thereby downregulated in differentiated cells via asymmetric division. MIIA is constitutive and activated by dephosphorylation during cytokine-triggered differentiation of cells grown on stiff, endosteum-like matrix, but not soft, marrow-like matrix. In vivo, MIIB is required for generation of blood, while MIIA is required for sustained HSC/P engraftment. Reversible inhibition of both isoforms in culture with blebbistatin enriches for long-term hematopoietic multilineage reconstituting cells by 5-fold or more as assessed in vivo. Megakaryocytes also become more polyploid, producing 4-fold more platelets. MII is thus a multifunctional node in polarized division and niche sensing.

Successful pregnancy requires coordination of an array of signals and factors from multiple tissues. One such element, liver receptor homolog-1 (Lrh-1), is an orphan nuclear receptor that regulates metabolism and hormone synthesis. It is strongly expressed in granulosa cells of ovarian follicles and in the corpus luteum of rodents and humans. Germline ablation of Nr5a2 (also called Lrh-1), the gene coding for Lrh-1, in mice is embryonically lethal at gastrulation. Depletion of Lrh-1 in the ovarian follicle shows that it regulates genes required for both steroid synthesis and ovulation. To study the effects of Lrh-1 on mouse gestation, we genetically disrupted its expression in the corpus luteum, resulting in luteal insufficiency. Hormone replacement permitted embryo implantation but was followed by gestational failure with impaired endometrial decidualization, compromised placental formation, fetal growth retardation and fetal death. Lrh-1 is also expressed in the mouse and human endometrium, and in a primary culture of human endometrial stromal cells, reduction of NR5A2 transcript abundance by RNA interference abrogated decidualization. These findings show that Lrh-1 is necessary for maintenance of the corpus luteum, for promotion of decidualization and for formation of the placenta. It therefore has multiple, indispensible roles in establishing and sustaining pregnancy.

Emerging data suggest that microRNAs (miRNAs) are instrumental in a variety of stress responses in addition to their more recognized role in development. Surprisingly, miRNAs, which normally suppress expression of target transcripts, may become activators of expression during stress. This might be partially explained by new interactions of miRNA/Argonaute complexes with RNA-binding proteins that relocate from different subcellular compartments during stress.


10% of sudden infant death syndrome (SIDS) deaths happen in newborns aged less than 6 months.

Gram-negative bacteria have an outer membrane (OM) that functions as a barrier to protect the cell from toxic compounds such as antibiotics and detergents. The OM is a highly asymmetric bilayer composed of phospholipids, glycolipids, and proteins. Assembly of this essential organelle occurs outside the cytoplasm in an environment that lacks obvious energy sources such as ATP, and the mechanisms involved are poorly understood. We describe the identification of a multiprotein complex required for the assembly of proteins in the OM of Escherichia coli. We also demonstrate genetic interactions between genes encoding components of this protein assembly complex and imp, which encodes a protein involved in the assembly of lipopolysaccharides (LPS) in the OM. These genetic interactions suggest a role for YfgL, one of the lipoprotein components of the protein assembly complex, in a homeostatic control mechanism that coordinates the overall OM assembly process.

CONTEXT Mesenchymal stem cells (MSCs) are under evaluation as a therapy for ischemic cardiomyopathy (ICM). Both autologous and allogeneic MSC therapies are possible; however, their safety and efficacy have not been compared. OBJECTIVE To test whether allogeneic MSCs are as safe and effective as autologous MSCs in patients with left ventricular (LV) dysfunction due to ICM. DESIGN, SETTING, AND PATIENTS A phase 1/2 randomized comparison (POSEIDON study) in a US tertiary-care referral hospital of allogeneic and autologous MSCs in 30 patients with LV dysfunction due to ICM between April 2, 2010, and September 14, 2011, with 13-month follow-up. INTERVENTION Twenty million, 100 million, or 200 million cells (5 patients in each cell type per dose level) were delivered by transendocardial stem cell injection into 10 LV sites. MAIN OUTCOME MEASURES Thirty-day postcatheterization incidence of predefined treatment-emergent serious adverse events (SAEs). Efficacy assessments included 6-minute walk test, exercise peak VO2, Minnesota Living with Heart Failure Questionnaire (MLHFQ), New York Heart Association class, LV volumes, ejection fraction (EF), early enhancement defect (EED; infarct size), and sphericity index. RESULTS Within 30 days, 1 patient in each group (treatment-emergent SAE rate, 6.7%) was hospitalized for heart failure, less than the prespecified stopping event rate of 25%. The 1-year incidence of SAEs was 33.3% (n = 5) in the allogeneic group and 53.3% (n = 8) in the autologous group (P = .46). At 1 year, there were no ventricular arrhythmia SAEs observed among allogeneic recipients compared with 4 patients (26.7%) in the autologous group (P = .10). Relative to baseline, autologous but not allogeneic MSC therapy was associated with an improvement in the 6-minute walk test and the MLHFQ score, but neither improved exercise VO2 max. Allogeneic and autologous MSCs reduced mean EED by −33.21% (95% CI, −43.61% to −22.81%; P < .001) and sphericity index but did not increase EF. Allogeneic MSCs reduced LV end-diastolic volumes. Low-dose concentration MSCs (20 million cells) produced greatest reductions in LV volumes and increased EF. Allogeneic MSCs did not stimulate significant donor-specific alloimmune reactions. CONCLUSIONS In this early-stage study of patients with ICM, transendocardial injection of allogeneic and autologous MSCs without a placebo control were both associated with low rates of treatment-emergent SAEs, including immunologic reactions. In aggregate, MSC injection favorably affected patient functional capacity, quality of life, and ventricular remodeling. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01087996.

Despite declines in prevalence during the past two decades, sudden infant death syndrome (SIDS) continues to be the leading cause of death for infants aged between 1 month and 1 year in developed countries. Behavioural risk factors identified in epidemiological studies include prone and side positions for infant sleep, smoke exposure, soft bedding and sleep surfaces, and overheating. Evidence also suggests that pacifier use at sleep time and room sharing without bed sharing are associated with decreased risk of SIDS. Although the cause of SIDS is unknown, immature cardiorespiratory autonomic control and failure of arousal responsiveness from sleep are important factors. Gene polymorphisms relating to serotonin transport and autonomic nervous system development might make affected infants more vulnerable to SIDS. Campaigns for risk reduction have helped to reduce SIDS incidence by 50-90%. However, to reduce the incidence even further, greater strides must be made in reducing prenatal smoke exposure and implementing other recommended infant care practices. Continued research is needed to identify the pathophysiological basis of SIDS.

Lymphocytes provide optimal responses against pathogens with minimal inflammatory pathology. However, the intrinsic mechanisms regulating these responses are unknown. Here, we report that deletion of both transcription factors Egr2 and Egr3 in lymphocytes resulted in a lethal autoimmune syndrome with excessive serum proinflammatory cytokines but also impaired antigen receptor-induced proliferation of B and T cells. Egr2- and Egr3-defective B and T cells had hyperactive signal transducer and activator of transcription-1 (STAT1) and STAT3 while antigen receptor-induced activation of transcription factor AP-1 was severely impaired. We discovered that Egr2 and/or Egr3 directly induced expression of suppressor of cytokine signaling-1 (SOCS1) and SOCS3, inhibitors of STAT1 and STAT3, and also blocked the function of Batf, an AP-1 inhibitor, in B and T cells. Thus, Egr2 and Egr3 regulate B and T cell function in adaptive immune responses and homeostasis by promoting antigen receptor signaling and controlling inflammation.


Pharmacist attendance at ward rounds reduces adverse events in wards.

Histone levels are tightly regulated to prevent harmful effects such as genomic instability and hypersensitivity to DNA-damaging agents due to the accumulation of these highly basic proteins when DNA replication slows down or stops. Although chromosomal histones are stable, excess (non-chromatin bound) histones are rapidly degraded in a Rad53 (radiation sensitive 53) kinase-dependent manner in Saccharomyces cerevisiae. Here we demonstrate that excess histones associate with Rad53 in vivo and seem to undergo modifications such as tyrosine phosphorylation and polyubiquitylation, before their proteolysis by the proteasome. We have identified the Tyr 99 residue of histone H3 as being critical for the efficient ubiquitylation and degradation of this histone. We have also identified the ubiquitin conjugating enzymes (E2) Ubc4 and Ubc5, as well as the ubiquitin ligase (E3) Tom1 (temperature dependent organization in mitotic nucleus 1), as enzymes involved in the ubiquitylation of excess histones. Regulated histone proteolysis has major implications for the maintenance of epigenetic marks on chromatin, genomic stability and the packaging of sperm DNA.

Transcription factors, such as Oct4, are critical for establishing and maintaining pluripotent cell identity. Whereas the genomic locations of several pluripotency transcription factors have been reported, the spectrum of their interaction partners is underexplored. Here, we use an improved affinity protocol to purify Oct4-interacting proteins from mouse embryonic stem cells (ESCs). Subsequent purification of Oct4 partners Sall4, Tcfcp2l1, Dax1, and Esrrb resulted in an Oct4 interactome of 166 proteins, including transcription factors and chromatin-modifying complexes with documented roles in self-renewal, but also many factors not previously associated with the ESC network. We find that Esrrb associated with the basal transcription machinery and also detect interactions between transcription factors and components of the TGF-beta, Notch, and Wnt signaling pathways. Acute depletion of Oct4 reduced binding of Tcfcp2l1, Dax1, and Esrrb to several target genes. In conclusion, our purification protocol allowed us to bring greater definition to the circuitry controlling pluripotent cell identity.

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.

EBI2, aka GPR183, is a G-couple receptor originally identified in 1993 as one of main genes induced in Burkitt's lymphoma cell line BL41 by Epstein-Barr virus (EBV) infection. After it was reported in 2009 that the receptor played a key role in regulating B cell migration and responses, we initiated an effort in looking for its endogenous ligand. In 2011 we and another group reported the identification of 7α, 25-dihydroxyxcholesterol (7α, 25-OHC), an oxysterol, as the likely physiological ligand of EBI2. A few subsequently published studies further elucidated how 7α, 25-OHC bound to EBI2, and how a gradient of 7α, 25-OHC could be generated in vivo and regulated migration, activation, and functions of B cells, T cells, dendritic cells (DCs), monocytes/macrophages, and astrocytes. The identification of 7α, 25-OHC as a G protein-coupled receptor ligand revealed a previously unknown signaling system of oxysterols, a class of molecules which exert profound biological functions. Dysregulation of the synthesis or functions of these molecules is believed to contribute to inflammation and autoimmune diseases, cardiovascular diseases, neurodegenerative diseases, cancer as well as metabolic diseases such as diabetes, obesity, and dyslipidemia. Therefore EBI2 may represent a promising target for therapeutic interventions for human diseases.

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

The tastes of sugars (sweet) and glutamate (umami) are thought to be detected by T1r receptors expressed in taste cells. Molecular genetics and heterologous expression implicate T1r2 plus T1r3 as a sweet-responsive receptor,and T1r1 plus T1r3,as well as a truncated form of the type 4 metabotropic glutamate receptor (taste-mGluR4),as umami-responsive receptors. Here,we show that mice lacking T1r3 showed no preference for artificial sweeteners and had diminished but not abolished behavioral and nerve responses to sugars and umami compounds. These results indicate that T1r3-independent sweet- and umami-responsive receptors and/or pathways exist in taste cells.


Question:
Tirasemtiv has no effect on cardiac muscle.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

The P446L mutant Drosophila importin-beta (P446L-imp-beta) has been reported to prohibit--in dominant negative fashion--nuclear envelope (NE) assembly. Along elucidating the mode of action of P446L-imp-beta we studied in vitro NE assembly on Sepharose beads. While Drosophila embryo extracts support NE assembly over Sepharose beads coated with Ran, NE assembly does not take place in extracts supplied with exogenous P446L-imp-beta. A NE also forms over importin-beta-coated beads. Surprisingly, when immobilized to Sepharose beads P446L-imp-beta as efficiently recruits NE vesicles as normal importin-beta. The discrepancy in behavior of cytoplasmic and bead-bound P446L-imp-beta appears to be related to icreased--as compared to normal importin-beta--microtubule (MT) binding ability of P446L-imp-beta. While wild-type importin-beta is able to bind MTs and the binding decreases upon RanGTP interaction, P446L-imp-beta cannot be removed from the MTs by RanGTP. P446L-imp-beta, like normal importin-beta, binds some types of the nucleoporins that have been known to be required for NE assembly at the end of mitosis. It appears that the inhibitory effect of P446L-imp-beta on NE assembly is caused by sequestering some of the nucleoporins required for NE assembly to the MTs.

Granulomas are the pathological hallmark of tuberculosis (TB). However, their function and mechanisms of formation remain poorly understood. To understand the role of granulomas in TB, we analyzed the proteomes of granulomas from subjects with tuberculosis in an unbiased manner. Using laser-capture microdissection, mass spectrometry and confocal microscopy, we generated detailed molecular maps of human granulomas. We found that the centers of granulomas have a pro-inflammatory environment that is characterized by the presence of antimicrobial peptides, reactive oxygen species and pro-inflammatory eicosanoids. Conversely, the tissue surrounding the caseum has a comparatively anti-inflammatory signature. These findings are consistent across a set of six human subjects and in rabbits. Although the balance between systemic pro- and anti-inflammatory signals is crucial to TB disease outcome, here we find that these signals are physically segregated within each granuloma. From the protein and lipid snapshots of human and rabbit lesions analyzed here, we hypothesize that the pathologic response to TB is shaped by the precise anatomical localization of these inflammatory pathways during the development of the granuloma.

CONTEXT Hyperhomocysteinemia is caused by genetic and lifestyle influences, including low intakes of folate and vitamin B6. However, prospective data relating intake of these vitamins to risk of coronary heart disease (CHD) are not available. OBJECTIVE To examine intakes of folate and vitamin B6 in relation to the incidence of nonfatal myocardial infarction (MI) and fatal CHD. DESIGN Prospective cohort study. SETTING AND PATIENTS In 1980, a total of 80082 women from the Nurses' Health Study with no previous history of cardiovascular disease, cancer, hypercholesterolemia, or diabetes completed a detailed food frequency questionnaire from which we derived usual intake of folate and vitamin B6. MAIN OUTCOME MEASURE Nonfatal MI and fatal CHD confirmed by World Health Organization criteria. RESULTS During 14 years of follow-up, we documented 658 incident cases of nonfatal MI and 281 cases of fatal CHD. After controlling for cardiovascular risk factors, including smoking and hypertension and intake of alcohol, fiber, vitamin E, and saturated, polyunsaturated, and trans fat, the relative risks (RRs) of CHD between extreme quintiles were 0.69 (95% confidence interval [CI], 0.55-0.87) for folate (median intake, 696 microg/d vs 158 microg/d) and 0.67 (95% CI, 0.53-0.85) for vitamin B6 (median intake, 4.6 mg/d vs 1.1 mg/d). Controlling for the same variables, the RR was 0.55 (95% CI, 0.41-0.74) among women in the highest quintile of both folate and vitamin B6 intake compared with the opposite extreme. Risk of CHD was reduced among women who regularly used multiple vitamins (RR=0.76; 95% CI, 0.65-0.90), the major source of folate and vitamin B6, and after excluding multiple vitamin users, among those with higher dietary intakes of folate and vitamin B6. In a subgroup analysis, compared with nondrinkers, the inverse association between a high-folate diet and CHD was strongest among women who consumed up to 1 alcoholic beverage per day (RR =0.69; 95% CI, 0.49-0.97) or more than 1 drink per day (RR=0.27; 95% CI, 0.13-0.58). CONCLUSION These results suggest that intake of folate and vitamin B6 above the current recommended dietary allowance may be important in the primary prevention of CHD among women.

It is unclear how the immune response in early life becomes appropriately stimulated to provide protection while also avoiding excessive activation as a result of diverse new antigens. T cells are integral to adaptive immunity; mouse studies indicate that tissue localization of T cell subsets is important for both protective immunity and immunoregulation. In humans, however, the early development and function of T cells in tissues remain unexplored. We present here an analysis of lymphoid and mucosal tissue T cells derived from pediatric organ donors in the first two years of life, as compared to adult organ donors, revealing early compartmentalization of T cell differentiation and regulation. Whereas adult tissues contain a predominance of memory T cells, in pediatric blood and tissues the main subset consists of naive recent thymic emigrants, with effector memory T cells (T(EM)) found only in the lungs and small intestine. Additionally, regulatory T (T(reg)) cells comprise a high proportion (30-40%) of CD4(+) T cells in pediatric tissues but are present at much lower frequencies (1-10%) in adult tissues. Pediatric tissue T(reg) cells suppress endogenous T cell activation, and early T cell functionality is confined to the mucosal sites that have the lowest T(reg):T(EM) cell ratios, which suggests control in situ of immune responses in early life.

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.


Question:
Immune complex triggered cell death leads to sequestration of oxidized mitochondrial DNA.

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.

Neutrophil extracellular traps (NETs) are implicated in autoimmunity, but how they are generated and their roles in sterile inflammation remain unclear. Ribonucleoprotein immune complexes (RNP ICs), inducers of NETosis, require mitochondrial reactive oxygen species (ROS) for maximal NET stimulation. After RNP IC stimulation of neutrophils, mitochondria become hypopolarized and translocate to the cell surface. Extracellular release of oxidized mitochondrial DNA is proinflammatory in vitro, and when this DNA is injected into mice, it stimulates type I interferon (IFN) signaling through a pathway dependent on the DNA sensor STING. Mitochondrial ROS are also necessary for spontaneous NETosis of low-density granulocytes from individuals with systemic lupus erythematosus. This was also observed in individuals with chronic granulomatous disease, who lack NADPH oxidase activity but still develop autoimmunity and type I IFN signatures. Mitochondrial ROS inhibition in vivo reduces disease severity and type I IFN responses in a mouse model of lupus. Together, these findings highlight a role for mitochondria in the generation not only of NETs but also of pro-inflammatory oxidized mitochondrial DNA in autoimmune diseases.

The tricyclic antidepressant, amitriptyline, is an effective drug for the treatment of chronic tension-type headache and for other chronic pain syndromes, but it is also effective in the prophylaxis of an episodic type of headache such as migraine. However, its efficacy in episodic tension-type headache has not yet been clarified. We compared the efficacy of amitriptyline (25 mg/day) in 82 nondepressed patients with either chronic or episodic tension-type headache in an open-label study. Amitriptyline significantly reduced (P < 0.05) frequency and duration of headache as well as analgesic consumption in chronic, but not in episodic, tension-type headache. Further placebo-controlled trials, possibly with higher doses of amitriptyline, might confirm if the different pattern of response to amitriptyline can be explained in terms of different involvement of central nociception and of peripheral myofascial factors in the chronic and in the episodic forms of tension-type headache.

Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints.

CONTEXT Use of antiretroviral drugs, including protease inhibitors, for treatment of human immunodeficiency virus (HIV) infection has been anecdotally associated with hepatotoxicity, particularly in persons coinfected with hepatitis C or B virus. OBJECTIVES To ascertain if incidence of severe hepatotoxicity during antiretroviral therapy is similar for all antiretroviral drug combinations, and to define the role of chronic viral hepatitis in its development. DESIGN Prospective cohort study. SETTING University-based urban HIV clinic. PATIENTS A total of 298 patients who were prescribed new antiretroviral therapies between January 1996 and January 1998, 211 (71%) of whom received protease inhibitors as part of combination therapy (median follow-up, 182 days) and 87 (29%) of whom received dual nucleoside analog regimens (median follow-up, 167 days). Chronic hepatitis C and B virus infection was present in 154 (52%) and 8 (2.7%) patients, respectively. MAIN OUTCOME MEASURE Severe hepatotoxicity, defined as a grade 3 or 4 change in levels of serum alanine aminotransferase and aspartate aminotransferase, evaluated before and during therapy. RESULTS Severe hepatotoxicity was observed in 31 (10.4%) of 298 patients (95% confidence interval [CI], 7.2%-14.4%). Ritonavir use was associated with a higher incidence of toxicity (30%; 95% CI, 17.9% -44.6%). However, no significant difference was detected in hepatotoxicity incidence in other treatment groups, ie, nucleoside analogs (5.7%; 95% CI, 1.2%-12.9%), nelfinavir (5.9%; 95% CI, 1.2%-16.2%), saquinavir (5.9%; 95% CI, 0.15%-28.7%), and indinavir(6.8%; 95% CI, 3.0%-13.1 %). Although chronicviral hepatitis was associated with an increased risk of severe hepatotoxicity among patients prescribed nonritonavir regimens (relative risk, 3.7; 95% CI, 1.0-11.8), most patients with chronic hepatitis C or B virus infection (88%) did not experience significant toxic effects. Rate of severe toxicity with use of any protease inhibitor in patients with hepatitis C infection was 12.2% (13/107; 95% CI, 6.6%-19.9%). In multivariate logistic regression, only ritonavir (adjusted odds ratio [AOR], 8.6; 95% CI, 3.0-24.6) and a CD4 cell count increase of more than 0.05 x 10(9)/L (AOR, 3.6; 95% CI, 1.0-12.9) were associated with severe hepatotoxicity. No irreversible outcomes were seen in patients with severe hepatotoxicity. CONCLUSIONS Our data indicate that use of ritonavir may increase risk of severe hepatotoxicity. Although hepatotoxicity may be more common in persons with chronic viral hepatitis, these data do not support withholding protease inhibitor therapy from persons coinfected with hepatitis B or C virus.


Question:
The microtubule-dependent delivery and secretion of matrix metalloproteases (MMPs) is partially responsible for the disassembly of adhesion sites.

Turnover of integrin-based focal adhesions (FAs) with the extracellular matrix (ECM) is essential for coordinated cell movement. In collectively migrating human keratinocytes, FAs assemble near the leading edge, grow and mature as a result of contractile forces and disassemble underneath the advancing cell body. We report that clustering of microtubule-associated CLASP1 and CLASP2 proteins around FAs temporally correlates with FA turnover. CLASPs and LL5β (also known as PHLDB2), which recruits CLASPs to FAs, facilitate FA disassembly. CLASPs are further required for FA-associated ECM degradation, and matrix metalloprotease inhibition slows FA disassembly similarly to CLASP or PHLDB2 (LL5β) depletion. Finally, CLASP-mediated microtubule tethering at FAs establishes an FA-directed transport pathway for delivery, docking and localized fusion of exocytic vesicles near FAs. We propose that CLASPs couple microtubule organization, vesicle transport and cell interactions with the ECM, establishing a local secretion pathway that facilitates FA turnover by severing cell-matrix connections.


Question:
Mouse models can be generated using "artificial spermatids."

Haploid cells are amenable for genetic analysis. Recent success in the derivation of mouse haploid embryonic stem cells (haESCs) via parthenogenesis has enabled genetic screening in mammalian cells. However, successful generation of live animals from these haESCs, which is needed to extend the genetic analysis to the organism level, has not been achieved. Here, we report the derivation of haESCs from androgenetic blastocysts. These cells, designated as AG-haESCs, partially maintain paternal imprints, express classical ESC pluripotency markers, and contribute to various tissues, including the germline, upon injection into diploid blastocysts. Strikingly, live mice can be obtained upon injection of AG-haESCs into MII oocytes, and these mice bear haESC-carried genetic traits and develop into fertile adults. Furthermore, gene targeting via homologous recombination is feasible in the AG-haESCs. Our results demonstrate that AG-haESCs can be used as a genetically tractable fertilization agent for the production of live animals via injection into oocytes.


A diminished ovarian reserve is a very strong indicator of infertility, even in an a priori non-infertile population.

Importance Despite lack of evidence of their utility, biomarkers of ovarian reserve are being promoted as potential markers of reproductive potential. Objective To determine the associations between biomarkers of ovarian reserve and reproductive potential among women of late reproductive age. Design, Setting, and Participants Prospective time-to-pregnancy cohort study (2008 to date of last follow-up in March 2016) of women (N = 981) aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, recruited from the community in the Raleigh-Durham, North Carolina, area. Exposures Early-follicular-phase serum level of antimüllerian hormone (AMH), follicle-stimulating hormone (FSH), and inhibin B and urinary level of FSH. Main Outcomes and Measures The primary outcomes were the cumulative probability of conception by 6 and 12 cycles of attempt and relative fecundability (probability of conception in a given menstrual cycle). Conception was defined as a positive pregnancy test result. Results A total of 750 women (mean age, 33.3 [SD, 3.2] years; 77% white; 36% overweight or obese) provided a blood and urine sample and were included in the analysis. After adjusting for age, body mass index, race, current smoking status, and recent hormonal contraceptive use, women with low AMH values (<0.7 ng/mL [n = 84]) did not have a significantly different predicted probability of conceiving by 6 cycles of attempt (65%; 95% CI, 50%-75%) compared with women (n = 579) with normal values (62%; 95% CI, 57%-66%) or by 12 cycles of attempt (84% [95% CI, 70%-91%] vs 75% [95% CI, 70%-79%], respectively). Women with high serum FSH values (>10 mIU/mL [n = 83]) did not have a significantly different predicted probability of conceiving after 6 cycles of attempt (63%; 95% CI, 50%-73%) compared with women (n = 654) with normal values (62%; 95% CI, 57%-66%) or after 12 cycles of attempt (82% [95% CI, 70%-89%] vs 75% [95% CI, 70%-78%], respectively). Women with high urinary FSH values (>11.5 mIU/mg creatinine [n = 69]) did not have a significantly different predicted probability of conceiving after 6 cycles of attempt (61%; 95% CI, 46%-74%) compared with women (n = 660) with normal values (62%; 95% CI, 58%-66%) or after 12 cycles of attempt (70% [95% CI, 54%-80%] vs 76% [95% CI, 72%-80%], respectively). Inhibin B levels (n = 737) were not associated with the probability of conceiving in a given cycle (hazard ratio per 1-pg/mL increase, 0.999; 95% CI, 0.997-1.001). Conclusions and Relevance Among women aged 30 to 44 years without a history of infertility who had been trying to conceive for 3 months or less, biomarkers indicating diminished ovarian reserve compared with normal ovarian reserve were not associated with reduced fertility. These findings do not support the use of urinary or blood follicle-stimulating hormone tests or antimüllerian hormone levels to assess natural fertility for women with these characteristics.


Incidence of 10/66 dementia is lower than the incidence of DSM-IV dementia.

BACKGROUND Results of the few cohort studies from countries with low incomes or middle incomes suggest a lower incidence of dementia than in high-income countries. We assessed incidence of dementia according to criteria from the 10/66 Dementia Research Group and Diagnostic and Statistical Manual of Mental Disorders (DSM) IV, the effect of dementia at baseline on mortality, and the independent effects of age, sex, socioeconomic position, and indicators of cognitive reserve. METHODS We did a population-based cohort study of all people aged 65 years and older living in urban sites in Cuba, the Dominican Republic, and Venezuela, and rural and urban sites in Peru, Mexico, and China, with ascertainment of incident 10/66 and DSM-IV dementia 3-5 years after cohort inception. We used questionnaires to obtain information about age in years, sex, educational level, literacy, occupational attainment, and number of household assets. We obtained information about mortality from all sites. For participants who had died, we interviewed a friend or relative to ascertain the likelihood that they had dementia before death. FINDINGS 12,887 participants were interviewed at baseline. 11,718 were free of dementia, of whom 8137 (69%) were reinterviewed, contributing 34,718 person-years of follow-up. Incidence for 10/66 dementia varied between 18·2 and 30·4 per 1000 person-years, and were 1·4-2·7 times higher than were those for DSM-IV dementia (9·9-15·7 per 1000 person-years). Mortality hazards were 1·56-5·69 times higher in individuals with dementia at baseline than in those who were dementia-free. Informant reports suggested a high incidence of dementia before death; overall incidence might be 4-19% higher if these data were included. 10/66 dementia incidence was independently associated with increased age (HR 1·67; 95% CI 1·56-1·79), female sex (0·72; 0·61-0·84), and low education (0·89; 0·81-0·97), but not with occupational attainment (1·04; 0·95-1·13). INTERPRETATION Our results provide supportive evidence for the cognitive reserve hypothesis, showing that in middle-income countries as in high-income countries, education, literacy, verbal fluency, and motor sequencing confer substantial protection against the onset of dementia. FUNDING Wellcome Trust Health Consequences of Population Change Programme, WHO, US Alzheimer's Association, FONACIT/ CDCH/ UCV.


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

The purpose of this study was to examine differences in resting, submaximal, and maximal (VO2max) oxygen consumption (VO2) in African-American (n = 44) and Caucasian (n = 31) prepubertal children aged 5-10 yr. Resting VO2 was measured via indirect calorimetry in the fasted state. Submaximal VO2 and VO2max were determined during an all out, progressive treadmill exercise test appropriate for children. Dual-energy X-ray absorptiometry was used to determine total fat mass (FM), soft lean tissue mass (LTM), and leg soft LTM. Doubly labeled water was used to determine total energy expenditure (TEE) and activity energy expenditure (AEE). A significant effect of ethnicity (P < 0.01) was found for VO2max but not resting or submaximal VO2, with African-American children having absolute VO2max approximately 15% lower than Caucasian children (1.21 +/- 0.032 vs. 1.43 +/- 0.031 l/min, respectively). The lower VO2max persisted in African-American children after adjustment for soft LTM (1.23 +/- 0.025 vs. 1.39 +/- 0.031 l/min; P < 0.01), leg soft LTM (1.20 +/- 0.031 vs. 1.43 +/- 0.042 l/min; P < 0.01), and soft LTM and FM (1.23 +/- 0.025 vs. 1.39 +/- 0.031 l/min; P < 0.01). The lower VO2max persisted also after adjustment for TEE (1.20 +/- 0.02 vs. 1.38 +/- 0.0028 l/min P < 0.001) and AEE (1.20 +/- 0.024 vs. 1.38 +/- 0.028 l/min; P < 0.001). In conclusion, our data indicate that African-American and Caucasian children have similar rates of VO2 at rest and during submaximal exercise, but VO2max is approximately 15% lower in African-American children, independent of soft LTM, FM, leg LTM, TEE, and AEE.

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

BACKGROUND Little is known about how the intensity of exercise influences cardiovascular fitness and body composition, especially in obese adolescents. OBJECTIVE Our goal was to determine the effects of physical training intensity on the cardiovascular fitness, percentage of body fat (%BF), and visceral adipose tissue (VAT) of obese adolescents. DESIGN Obese 13-16-y-olds (n = 80) were assigned to 1) biweekly lifestyle education (LSE), 2) LSE + moderate-intensity physical training, or 3) LSE + high-intensity physical training. The intervention lasted 8 mo. Physical training was offered 5 d/wk, and the target energy expenditure for all subjects in physical training groups was 1047 kJ (250 kcal)/session. Cardiovascular fitness was measured with a multistage treadmill test, %BF with dual-energy X-ray absorptiometry, and VAT with magnetic resonance imaging. RESULTS The increase in cardiovascular fitness in the high-intensity physical training group, but not in the moderate-intensity group, was significantly greater than that in the LSE alone group (P = 0.009); no other comparisons of the 3 groups were significant. Compared with the LSE alone group, a group composed of subjects in both physical training groups combined who attended training sessions >or=2 d/wk showed favorable changes in cardiovascular fitness (P < 0.001), %BF (P = 0.001), and VAT (P = 0.029). We found no evidence that the high-intensity physical training was more effective than the moderate-intensity physical training in enhancing body composition. CONCLUSIONS The cardiovascular fitness of obese adolescents was significantly improved by physical training, especially high-intensity physical training. The physical training also reduced both visceral and total-body adiposity, but there was no clear effect of the intensity of physical training.

CONTEXT Physical inactivity contributes to weight gain in adults, but whether this relationship is true for children of different ethnic groups is not well established. OBJECTIVE To assess participation in vigorous activity and television watching habits and their relationship to body weight and fatness in US children. DESIGN Nationally representative cross-sectional survey with an in-person interview and medical examination. SETTING AND PARTICIPANTS Between 1988 and 1994, 4063 children aged 8 through 16 years were examined as part of the National Health and Nutrition Examination Survey III. Mexican Americans and non-Hispanic blacks were oversampled to produce reliable estimates for these groups. MAIN OUTCOME MEASURES Episodes of weekly vigorous activity and daily hours of television watched, and their relationship to body mass index and body fatness. RESULTS Eighty percent of US children reported performing 3 or more bouts of vigorous activity each week. This rate was lower in non-Hispanic black and Mexican American girls (69% and 73%, respectively). Twenty percent of US children participated in 2 or fewer bouts of vigorous activity perweek, and the rate was higher in girls (26%) than in boys (17%). Overall, 26% of US children watched 4 or more hours of television per day and 67% watched at least 2 hours per day. Non-Hispanic black children had the highest rates of watching 4 or more hours of television per day (42%). Boys and girls who watch 4 or more hours of television each day had greater body fat (P<.001) and had a greater body mass index (P<.001) than those who watched less than 2 hours per day. CONCLUSIONS Many US children watch a great deal of television and are inadequately vigorously active. Vigorous activity levels are lowest among girls, non-Hispanic blacks, and Mexican Americans. Intervention strategies to promote lifelong physical activity among US children are needed to stem the adverse health consequences of inactivity.


Klf4 is not required for proper myeloid cell differentiation.

IMPORTANCE The programmed death 1 (PD-1) pathway limits immune responses to melanoma and can be blocked with the humanized anti-PD-1 monoclonal antibody pembrolizumab. OBJECTIVE To characterize the association of pembrolizumab with tumor response and overall survival among patients with advanced melanoma. DESIGN, SETTINGS, AND PARTICIPANTS Open-label, multicohort, phase 1b clinical trials (enrollment, December 2011-September 2013). Median duration of follow-up was 21 months. The study was performed in academic medical centers in Australia, Canada, France, and the United States. Eligible patients were aged 18 years and older and had advanced or metastatic melanoma. Data were pooled from 655 enrolled patients (135 from a nonrandomized cohort [n = 87 ipilimumab naive; n = 48 ipilimumab treated] and 520 from randomized cohorts [n = 226 ipilimumab naive; n = 294 ipilimumab treated]). Cutoff dates were April 18, 2014, for safety analyses and October 18, 2014, for efficacy analyses. EXPOSURES Pembrolizumab 10 mg/kg every 2 weeks, 10 mg/kg every 3 weeks, or 2 mg/kg every 3 weeks continued until disease progression, intolerable toxicity, or investigator decision. MAIN OUTCOMES AND MEASURES The primary end point was confirmed objective response rate (best overall response of complete response or partial response) in patients with measurable disease at baseline per independent central review. Secondary end points included toxicity, duration of response, progression-free survival, and overall survival. RESULTS Among the 655 patients (median [range] age, 61 [18-94] years; 405 [62%] men), 581 had measurable disease at baseline. An objective response was reported in 194 of 581 patients (33% [95% CI, 30%-37%]) and in 60 of 133 treatment-naive patients (45% [95% CI, 36% to 54%]). Overall, 74% (152/205) of responses were ongoing at the time of data cutoff; 44% (90/205) of patients had response duration for at least 1 year and 79% (162/205) had response duration for at least 6 months. Twelve-month progression-free survival rates were 35% (95% CI, 31%-39%) in the total population and 52% (95% CI, 43%-60%) among treatment-naive patients. Median overall survival in the total population was 23 months (95% CI, 20-29) with a 12-month survival rate of 66% (95% CI, 62%-69%) and a 24-month survival rate of 49% (95% CI, 44%-53%). In treatment-naive patients, median overall survival was 31 months (95% CI, 24 to not reached) with a 12-month survival rate of 73% (95% CI, 65%-79%) and a 24-month survival rate of 60% (95% CI, 51%-68%). Ninety-two of 655 patients (14%) experienced at least 1 treatment-related grade 3 or 4 adverse event (AE) and 27 of 655 (4%) patients discontinued treatment because of a treatment-related AE. Treatment-related serious AEs were reported in 59 patients (9%). There were no drug-related deaths. CONCLUSIONS AND RELEVANCE Among patients with advanced melanoma, pembrolizumab administration was associated with an overall objective response rate of 33%, 12-month progression-free survival rate of 35%, and median overall survival of 23 months; grade 3 or 4 treatment-related AEs occurred in 14%. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT01295827.

Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs-such as LINC00173 in granulocytes-and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy. While micro-RNAs are known regulators of haematopoiesis and leukemogenesis, the role of long non-coding RNAs is less clear. Here the authors provide a non-coding RNA expression landscape of the human hematopoietic system, highlighting their role in the formation and maintenance of the human blood hierarchy.

Several members of the Kruppel-like factor (KLF) family of transcription factors play important roles in differentiation, survival, and trafficking of blood and immune cell types. We demonstrate in this study that hematopoietic cells from KLF4(-/-) fetal livers (FL) contained normal numbers of functional hematopoietic progenitor cells, were radioprotective, and performed as well as KLF4(+/+) cells in competitive repopulation assays. However, hematopoietic "KLF4(-/-) chimeras" generated by transplantation of KLF4(-/-) fetal livers cells into lethally irradiated wild-type mice completely lacked circulating inflammatory (CD115(+)Gr1(+)) monocytes, and had reduced numbers of resident (CD115(+)Gr1(-)) monocytes. Although the numbers and function of peritoneal macrophages were normal in KLF4(-/-) chimeras, bone marrow monocytic cells from KLF4(-/-) chimeras expressed lower levels of key trafficking molecules and were more apoptotic. Thus, our in vivo loss-of-function studies demonstrate that KLF4, previously shown to mediate proinflammatory signaling in human macrophages in vitro, is essential for differentiation of mouse inflammatory monocytes, and is involved in the differentiation of resident monocytes. In addition, inducible expression of KLF4 in the HL60 human acute myeloid leukemia cell line stimulated monocytic differentiation and enhanced 12-O-tetradecanoylphorbol 13-acetate induced macrophage differentiation, but blocked all-trans-retinoic acid induced granulocytic differentiation of HL60 cells. The inflammation-selective effects of loss-of-KLF4 and the gain-of-KLF4-induced monocytic differentiation in HL60 cells identify KLF4 as a key regulator of monocytic differentiation and a potential target for translational immune modulation.

The unique metabolic demands of cancer cells underscore potentially fruitful opportunities for drug discovery in the era of precision medicine. However, therapeutic targeting of cancer metabolism has led to surprisingly few new drugs to date. The neutral amino acid glutamine serves as a key intermediate in numerous metabolic processes leveraged by cancer cells, including biosynthesis, cell signaling, and oxidative protection. Herein we report the preclinical development of V-9302, a competitive small molecule antagonist of transmembrane glutamine flux that selectively and potently targets the amino acid transporter ASCT2. Pharmacological blockade of ASCT2 with V-9302 resulted in attenuated cancer cell growth and proliferation, increased cell death, and increased oxidative stress, which collectively contributed to antitumor responses in vitro and in vivo. This is the first study, to our knowledge, to demonstrate the utility of a pharmacological inhibitor of glutamine transport in oncology, representing a new class of targeted therapy and laying a framework for paradigm-shifting therapies targeting cancer cell metabolism.

Ankylosing spondylitis (AS) is a common, highly heritable, inflammatory arthritis for which HLA-B*27 is the major genetic risk factor, although its role in the aetiology of AS remains elusive. To better understand the genetic basis of the MHC susceptibility loci, we genotyped 7,264 MHC SNPs in 22,647 AS cases and controls of European descent. We impute SNPs, classical HLA alleles and amino-acid residues within HLA proteins, and tested these for association to AS status. Here we show that in addition to effects due to HLA-B*27 alleles, several other HLA-B alleles also affect susceptibility. After controlling for the associated haplotypes in HLA-B, we observe independent associations with variants in the HLA-A, HLA-DPB1 and HLA-DRB1 loci. We also demonstrate that the ERAP1 SNP rs30187 association is not restricted only to carriers of HLA-B*27 but also found in HLA-B*40:01 carriers independently of HLA-B*27 genotype.